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DNA DAMAGE CAUSED

BY NORMAL METABOLIC

ACTIVITIES AND ENVI-

RONMENTAL FACTORS TRIGGERS THE

DNA DAMAGE RESPONSE SYSTEM TO

ACTIVATE A DNA REPAIR PATHWAY, OR IN

THE CASE OF IRREPARABLE DAMAGE,

INDUCES APOPTOSIS.

MUTATIONS IN THE

GENES THAT ENCODE

DNA DAMAGE RESPONSE PROTEINS

MAY RESULT IN GENOMIC INSTABILITY.

PROGRESSIVE GENOMIC INSTABILITY IS

A HALLMARK OF CANCER

PROGRESSION AND MU-

TATION IN DNA DAMAGE

RESPONSE PROTEINS AND MAY BE A GEN-

ERAL FEATURE OF CANCER. EPIGENETIC

SILENCING OF GENES HAS ALSO BEEN

IMPLICATED IN THE PATHOGENESIS OF

CANCER. CANCER CELL

BEHAVIORS AND THE

TOOLS REQUIRED TO

STUDY THEIR MANY FACETS, INCLUDING

CELL MIGRATION, CELL INVASION, CELL

ADHESION AND CELL DIFFERENTIATION

ARE ALL A FOCUS OF

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HOW TO ORDER

Please use product names and catalog numbers when ordering. If ordering from a quotation, please include quote number and the lot number for reserved lot inventory. Please provide your name, purchase order number, shipping address, billing address, date, and telephone number on each order.

Trevigen accepts VISA, MASTERCARD, and AMERICAN EXPRESS.

Fax, phone, e-mail, or mail your order to:

Trevigen, Inc. 8405 Helgerman Court Gaithersburg, MD 20877 1-800-873-8443 Local Phone: 301-216-2800 Fax: 301-560-4973 E-mail: [email protected]

Order online at www.trevigen.com.

International Distributors – see inside back cover.

SHIPMENTS: All shipments are made through our carrier unless otherwise requested. Shipping charges are prepaid and added to your invoice. For RUSH orders, please advise the Trevigen’s Customer Service Representative at the time of order.

TERMS: Payment should be made to Trevigen, Inc., P.O. Box 7328, Gaithersburg, MD 20898, within thirty (30) days of shipment, in U.S. dollars.

CONDITIONS: Trevigen products are sold for Research Use Only.

PRODUCT USAGE: Products manufactured and sold by Trevigen are for the customer’s research purposes only. Resale of Trevigen products requires the express written consent of Trevigen. Trevigen products have not been approved for use in any clinical, diagnostic, or therapeutic applications. Obtaining license or approval to use Trevigen products in proprietary applications or in any non-research (clinical) applications is the customer’s exclusive responsibility. Trevigen will not be responsible or liable for any losses, costs, expenses, or liability arising out of the unauthorized or unlicensed use of Trevigen products.

WARRANTY: Trevigen warrants that products will perform according to specifications accompanying each product. Products found not meeting specifications will be replaced, free of charge. Trevigen’s liability is limited to replacement of the product. Notice for replacement must be given within 90 days of receipt of product. Shipping discrepancies must be reported within 5 business days of receipt of product. All other warranties are hereby expressly disclaimed, including but not limited to, the implied warranties of merchantability and fitness for a particular purpose, and any warranty that the products, or the use of products, manufactured by Trevigen will not infringe the patents of one or more third parties. Trevigen shall not be liable for any consequential damages. Discrepancies with items ordered through a Trevigen distributor must be handled with the distributor.

OTHER: Prices are subject to change. Trevigen reserves the right to change product specifications. Products may vary somewhat from the catalog description.

TRADEMARKS: CometAssay, CultreCoat, Cultrex, PathClear, TACS, TACS•XL, and Trevigen, are registered trademarks, and 3-D Matrix, Apoptosis Grade, CardioTACS, CometSlide, Cytonin, Cytonin IHC, DePsipher, DermaTACS, DIVAA, FLARE, FlowTACS, MitoShift, NeuroPore, NeuroTACS, PeroxyGlow, REC, TACS Blue Label, TACS-Nuclease, TACS-Sapphire, TiterTACS, TreviGel, TumorTACS, and VasoTACS are trademarks of Trevigen, Inc.

EpiDerm is a trademark of MatTek Corporation, Ashland, MA. SYBR Green I is a registered trademark of Molecular Probes, Inc., Eugene, OR. Tween 20 is a registered trademark of ICI Americas, Inc., Wilmington, DE. The PCR process is protected by patents owned by Hoffmann-LaRoche.

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OVERVIEW

Overview

Dear Researcher,

Our 2011/2012 catalog reflects Trevigen’s mission: “To provide cancer researchers with the highest quality products and services for ultimate benefit toward the prevention and cure of cancer.” Thus, we have developed products that focus on key causative aspects emanating from DNA damage, DNA repair, and genomic instability, as well as products to detect and quantitate the downstream impact on cancer cell behavior.

Over the past few years, our specialized products for the characterization of cancer cell behavior have found numerous research applications in the areas of:

• Screening Therapeutics for Cancer Treatments

• Conducting Pharmacodynamic Assays on Lead Compounds

• Tumor Explant Work

• Cell Adhesion

• Metastasis

• Angiogenesis

• 3-D Structure Studies

• Apoptosis Detection

Acknowledging the complexity inherent in cancer cells and therefore cancer cell assays, Trevigen maintains a technical support team to assist you, the researcher, in assay selection, set up, troubleshooting as well as the interpretation of results.

We take pride in the quality of our products, which is matched by the fast turnaround and thoughtful responses of both the technical support and customer service teams.Our goal is to make sure you know you are able to “count on us”, at each step of the research process, from the planning stages of your project, to data analysis/interpretation.

We organized our new catalog with our customers in mind; to make it easier for you to locate, find specifications, and to order our products.

We also strive to keep our website up-to-date. As new products are released and new applications information becomes available for old products, we make sure the information is available immediately on our website.

Please do not hesitate to contact us at 800-873-8443 or [email protected] if you have any questions at all.

On behalf of all of us at Trevigen, Inc., I wish you continued success in your research.

Sincerely,

Michael T. Elliott

President, Trevigen, Inc.

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Table of Contents

How To Order ...... i • Human FEN‐1 (Flap Endonuclease) ...... 47 Overview ...... ii • hOGG1 DNA Glycosylase ...... 48 • E. Coli Endonuclease III ...... 49 DNA DAMAGE AND GENOMIC INSTABILITY • E. Coli Fpg DNA Glycosylase ...... 50 • Overview ...... 4 • S. pombe UVDE ...... 51 DNA Repair Gene Knockdown Cell Lines ...... 5 • T4 Endonuclease V ...... 52 PARP/PAR/PARG • Cv‐PDG ...... 54 • PARP Selection Guide ...... 7 • E. Coli Endonuclease IV ...... 56 • PARP and PARG Selection Guide ...... 8 • UNGase DNA Glycosylase ...... 58 • Universal PARP Assay Kits ...... 9 • Aag DNA Glycosylase ...... 60 • PARP/Apoptosis Assay Kits ...... 10 • Mug DNA Glycosylase ...... 61 • Fluorescent Homogeneous PARP Inhibition Assay . . .11 • MutY DNA Glycosylase ...... 62 • PARP In Vivo Pharmacodynamic Assay II ...... 12 • TDG DNA Glycosylase ...... 63 • Universal PARG Assay Kits ...... 14 • Dpo4 Polymerase ...... 64 • PARP Enzyme ...... 15 • Human Ku 70/80 Complex ...... 65 • PARG Enzyme ...... 16 • PAR Polymer ...... 17 CANCER CELL BEHAVIOR • PARP Monoclonal Antibody ...... 18 • Overview/ Cultrex® Product Selection Guide ...... 68 • PAR Monoclonal Antibody ...... 19 Basement Membrane Extract (BME), Mouse ...... 70 • PAR Polyclonal Antibody ...... 20 • BME Selection Guide ...... 71 • Biotin‐NAD⁺ ...... 21 • BME PathClear® ...... 72 • Fluorescein‐NAD⁺ ...... 22 • BME High Protein Concentration ...... 74 • Cell Permeabilization Solution ...... 22 • BME Stem Cell Qualified ...... 75 • PARP and PARG Inhibitors ...... 23 • BME 3‐D Culture Matrix™ ...... 76 CometAssay™ ECM Component Proteins and Reagents • Reagent Kits for Single Cell Gel Electrophoresis Assay .24 • Overview/ Selection Guide ...... 77 • Silver Staining Kit ...... 26 • Human BME ...... 80 • Electrophoresis System ...... 27 • Human Fibronectin ...... 81 • Control Cells ...... 29 • Human Vitronectin ...... 82 • Slides and Rack System ...... 30 • Mouse Laminin I, ...... 83 • FLARE™ Kits and Slides/ Selection Guide ...... 31 • Mouse Laminin I PathClear® ...... 84 • Fpg Assay Kit and Module ...... 32 • Rat Collagen I ...... 85 • hOGGI Assay Kit and Module ...... 33 • Mouse Collagen IV ...... 86 • Endonuclease III Assay Kit and Module ...... 34 • Bovine Collagen I ...... 87 • UVDE Assay Kit and Module ...... 35 • Bovine Fibronectin ...... 88 • T4‐PDG Assay Kit and Module ...... 36 • Bovine Vitronectin ...... 89 • cv‐PDG Assay Kit and Module ...... 37 • Poly‐D‐Lysine ...... 90 DNA Damage Antibodies • Poly‐L‐Lysine ...... 91 • Selection Guide ...... 38 • 3‐D Culture Matrix™ Mouse Laminin ...... 92 • γ‐H2AX ...... 39 • 3‐D Culture Matrix™ Rat Collagen I ...... 93 • 8‐oxo‐dG ...... 40 Cancer Cell Assays • FEN-1 ...... 41 • Overview/ Selection Guide ...... 94 • BPDE ...... 42 • Directed In Vivo Angiogenesis Assay (DIVAA™) . . . . .95 • UVssDNA ...... 43 • In Vitro Angiogenesis Assay Tube Formation Kit . . . . .97 DNA Repair Enzymes • In Vitro Angiogenesis Assay Endothelial Cell • Selection Guide ...... 44 Invasion Kit ...... 98 • hAPE ...... 45 • CultreCoat® Vascular Permeability Assays ...... 99 • Human DNA Polymerase β kit ...... 46 • Cell Invasion Assays ...... 100

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Table of Contents

• Cell Invasion Optimization System ...... 100 STEM CELL PRODUCTS • Cell Migration Assays ...... 100 • Overview ...... 164 • CultreCoat® Cell Adhesion Assays ...... 102 • Stem Cell Qualified BME ...... 165 • Cell Staining Kit ...... 104 • Rat Mesenchymal Stem Cell Growth 3‐D Products and Replenishment products ...... 166 • 3‐D Culture Matrix™ BME ...... 105 • Mesenchymal Stem Cell Adipogenic • 3‐D Culture Matrix™ Mouse Laminin I ...... 106 Differentiation Kit ...... 167 • 3‐D Culture Matrix™ Rat Tail Collagen I ...... 107 • Mesenchymal Stem Cell Osteogenic • 3‐D Proliferation Assays ...... 108 Differentiation Kit ...... 168 • 3‐D Culture Cell Harvesting Kit ...... 109 • MTT/XTT Cell Proliferation Assay ...... 111 OXIDATIVE STRESS • Calcein‐AM Cell Viability Assay ...... 111 • Overview/ Selection Guide ...... 170 • 8‐oxo‐dG ELISA Kit ...... 171 APOPTOSIS DETECTION • 8‐oxo‐dG Monoclonal Antibody ...... 172 • Overview/ Selection Guide ...... 114 • HT Superoxide Dismutase Assay Kit ...... 173 Annexin V Assays • Superoxide Dismutase Assay Kit ...... 174 • TACS® Annexin V‐FITC Kit ...... 118 • HT Glutathione Assay Kit ...... 175 • TACS® Annexin V‐Biotin Kit ...... 120 • HT Glutathione Peroxide Assay Kit ...... 176 DNA Fragmentation • HT Glutathione Reductase Assay Kit ...... 177 • TACS.XL® In Situ Apoptosis Detection Kits ...... 121 • Glutathione Reductase Assay Kit ...... 178 • TACS® 2TdT In Situ Apoptosis Detection Kits ...... 123 • Specialty In Situ Apoptosis Detection/ MOLECULAR BIOLOGY REAGENTS Selection Guide ...... 125 • Overview ...... 180 • CardioTACS™ ...... 126 • Genomic DNA Isolation Kit ...... 181 • DermaTACS™ ...... 128 • TreviGel™ ...... 182 • NeuroTACS™ ...... 130 • Orange G Loading Dye ...... 183 • TumorTACS™ ...... 132 • Electrophoresis Buffer ...... 183 • VascoTACS™ ...... 134 • PBS ...... 183 • TiterTACS™ ...... 135 • TdT DNA Polymerase ...... 184 • FlowTACS™ ...... 137 • Calf Thymus DNA, Herring Sperm DNA, • TACS® DNA Laddering Kit ...... 139 • Salmon Sperm DNA ...... 184 Mitochondrial Events • Proteinase K ...... 184 • DePsipher™ ...... 140 • MitoShift™ ...... 142 TREVIGEN CELL ASSAYS (TCA) SERVICES • PARP/Apoptosis Assay Kits ...... 144 • CometAssay™ ...... 186 Cell Proliferation • PARP Assay ...... 186 • TACS® MTT Cell Proliferation Assay ...... 145 • PARG Assay ...... 186 • TACS® XTT Cell Proliferation Assay ...... 146 • Pharmacodynamic PARP Assay ...... 186 • Calcein AM Cell Viability Kit ...... 147 • Cell Invasion Assay ...... 186 Antibodies • Tube Formation Assay ...... 186 • Selection Guide ...... 148 • Bax ...... 149 Price List ...... 187 • Bcl‐2 ...... 151 Appendix ...... 192 • Bcl‐XL ...... 152 Index ...... 193 • PBR Antibody and Protein ...... 153 Technical Resources ...... 195 • Cleaved Caspase‐3 ...... 154 International Distributors ...... 198 • G3PDH ...... 155 Apoptosis Research Accessories ...... 156 Apoptosis Product Use ...... 159

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DNA DAMAGE AND GENOMIC INSTABILITY

Cancer Cell Behavior

Apoptosis Detection

Stem Cell Products

Oxidative Stress

Molecular Biology Reagents DNA DAMAGE AND GENOMIC INSTABILITY DNA DAMAGE INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:11 PM Page 8

DNA DAMAGE AND GENOMIC INSTABILITY

Overview

Trevigen is pleased to provide highly qualified enzymes, antibodies, and assay kits for DNA damage and repair research. In this catalog, we have NEW and UNIQUE additions to our product line to complement and provide your research efforts with flexibility. As always, our products are accompanied by detailed product data sheets or instructions for use backed with expert technical support.

DNA REPAIR DEFICIENT CELL LINES DNA repair pathways maintain the integrity of the genome reducing the onset of cancer, disease and aging phenotypes. However, the requirement for DNA repair and genome maintenance in response to radiation and genotoxic chemotherapies implicates DNA repair proteins as prime targets for improving response to currently available anti-cancer approaches. To further study repair pathways, Trevigen offers panels of human cell lines each deficient in proteins associated with DNA repair. The first panel consists of cell lines deficient for proteins in the Base Excision Repair Pathway. For the availability of other DNA repair deficient cell lines contact Trevigen.

PARP/PAR/PARG PARP-1 contributes to the sequence of events that occurs during DNA base excision repair. Poly (ADP-ribose) polymerase (PARP) catalyzes the NAD+ - dependant addition of poly (ADP-ribose) (PAR) onto itself and adjacent nuclear proteins in response to DNA damage.

Since PAR is often highly branched and degraded by poly(ADP-ribose) glycohydrolase DNA DAMAGE AND GENOMIC INSTABILITY OVERVIEW (PARG), Trevigen offers kits that measure the in vivo and in vitro activities of PARP and PARG. One hallmark of apoptosis is caspase-mediated cleavage and inactivation of PARP-1. 4 The PARP Apoptosis Kits measure decreasing levels of PARP activity as cells move through the apoptosis pathway. The Homogeneous PARP Inhibition Assay is well suited for the large scale screening of compound libraries for candidate PARP inhibitors. Our Universal PARP and PARG Assays are designed to analyze small numbers of inhibitors and recommended

for providing accurate IC50 information. Inhibitors to PARP can be characterized by in vitro assays. Their in vivo activity in cell or tissue extracts, or peripheral blood mononuclear cells can be quantitatively measured using Trevigen’s validated PARP in vivo Pharmacodynamic Assay. Biotinylated NAD+, PAR and PARP antibodies, key reagents for the described assays, are also available as individual components.

COMETASSAY® The single cell gel electrophoresis or CometAssay® is a state-of-the-art technique for quantitating DNA damage and repair from in vivo and in vitro samples of eukaryotic cells and some prokaryotic cells. In conjunction with Trevigen’s new CometAssay® Electrophoresis System, which eliminates known causes of assay variability, it is the only technique that directly measures DNA damage in individual cells and as a result has rapidly gained importance in the fields of genetic toxicology and human biomonitoring. Trevigen’s CometAssay® measures double strand breaks (DSBs), single strand breaks (SSBs), alkali labile sites, oxidative DNA base damage, DNA-DNA/ DNA-protein/DNA-Drug crosslinking and DNA repair.

DNA REPAIR ENZYMES AND ANTIBODIES Trevigen’s unique FLARE™ Assays characterize DNA damage in single cells using a variety of DNA repair enzymes that recognize oxidative and UV damage in conjunction with Trevigen’s CometAssay® Electrophoresis System. A variety of DNA repair enzymes and antibodies are available as individual components with optimized assay conditions and buffers for research in areas of double-strand break repair, base excision repair, DNA methylation, oxidative damage, UV damage, and radiation damage.

Please check our website for the latest product additions and product information.

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Gene Knockdown Cell Lines Tools to Study Genomic Instability and Genotoxic Stress DNA Repair Gene Knockdown Cell Lines* Base Excision Repair

DNA repair proteins maintain the stability of the genome. When repair protein function is impaired through mutations the genome may become unstable, which is a hallmark of solid tumors. The availability of this panel of knockdown cell lines will permit scientists to study the molecular etiology of tumor genomic instability and to exploit it in oncology research. The first panel covers the Base Excision Repair pathway.

Trevigen’s Base Excision Repair Knockdown Cell Lines are target specific LN428 (glioblas- KNOCKDOWN CELL LINES toma) shRNA lentivirus transduced cells. They are rigorously qualified and mycoplasma free. The percent knockdown levels range from 63% -98% as evaluated by RT-PCR. Lentiviruses are maintained by puromycin selection.

FEATURES: OVERVIEW Tested negative for Mycoplasma

Tested negative for: Human immunodeficiency virus (HIV1, HIV2), Hepatitis viruses (A, B, and C), Human T-lymphotropic virus (HTLV 1, HTLV 2), Epstein Barr, Hantaviruses Hantaan (Seoul, Sin, Nombre), Herpes simplex (1+2,) Human cytomegalovirus, Human herpes virus (6+8), Human adenovirus, Varicella virus and Lymphocytic Choriomeningitis virus.

ORDERING INFORMATION: 005 * Cell lines are provided under an MTA. Please inquire at [email protected]

¹BER-Base Excision Repair ²HR- Homologous Recombination ³LN428- Human malignant glioblastoma cell line *BRCA1 participates in the Homologous Recombination Pathway and demonstrates synthetic lethality in combination with PARP1.

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Gene Knockdown Cell Lines BRCA1 Knockdown cell line is sensitive to PARP1 inhibitors

All Trevigen knockdown cell lines are evaluated by RT-PCR and Western blots when reliable immunoreagents are available. Additional functional assays are performed when feasible.

Enzymatic Assay for MPG activity

Lane 1: Buffer alone Knockdown cell line growth in presence of PARP inhibitor. Lane 2: Control extract KNOCKDOWN CELL LINES Lane 3: MPG knockdown REFERENCES: 6 1. Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA, Ellenberger T: DNA Repair and Mutagenesis, 2nd Edition. Washington, D.C.: ASM Press; 2006. 2. Alberts B: Redefining cancer research. Science 2009, 325(5946):1319 (PMID:19745119). 3. Ljungman M: Targeting the DNA damage response in cancer. Chem Rev 2009, Western Blot of MPG protein 109(7):2929-2950 (PMID:19545147). 4. Peralta-Leal A, Rodriguez MI, Oliver FJ: Poly(ADP-ribose)polymerase-1 (PARP-1) in carcinogenesis: potential role of PARP inhibitors in cancer treatment. Clin Transl Oncol 2008, 10(6):318-323 (PMID:18558578).

STORAGE: Liquid Nitrogen

LIMITED USE NOTICE: Trevigen’s DNA Repair Gene Knock Down Cell Lines can only be obtained under a Materials Transfer Agreement executed with Trevigen, Inc. The Materials Transfer Agreement conveys Lane 1: Molecular Weight Markers to the buyer the non-transferable right to use the purchased amount of the product and all Lane 2: Scramble RNA Control replicates and derivatives for research purposes conducted by the buyer, in their laboratory only. For details on the Materials Transfer Agreement or for information on purchasing a Lane 3: MPG knockdown extract license for these products for purposes other than research, contact Trevigen, Inc. Lane 4: Control cell line

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG hibitors PARP Assay Selection Guide

High Throughput Screening for PARP Inhibitors HT F Homogeneous PARP PARP Homogeneous Assay Inhibition Assay Kit # 4690-096-K

poly-ADP ribose Step 2: Cycling Assay

PARP 1 AD PARP ASSAY SELECTION GUIDE inactive EtOH Acetaldehyde

nicotinamide [NAD+] NADH +H

Activated + PARP 1 Verification of Lead Compounds or DNA active Diaphorase Limited Inhibitor Screening Campaigns. Step 1: PARP Reaction Determination of IC Values 50 OVERVIEW Fluorescent Resorufin Resazurin hv ~ [NAD+] remaining after Step 1

HT Universal PARP Assay Kits Trevigen’s # 4677-096-K Colorimetric Universal PARP Assay # 4676-096-K Chemiluminescent

Colorimetric or Chemiluminescent 007 Detection

HRP

Streptavidin

Biotin Determine PARP Activity Evaluation of Inhibitor Behavior In Vivo or in Cultured Cells in Cell Lysates PAR HT PARP/ Apoptosis Assay Kit # 4684-096-K Colorimetric # 4685-096-K Chemiluminescent Histones

Trevigen’s PARP Trevigen’s PARP Apoptosis Assay Pharmacodynamic Assay

Colorimetric or Chemiluminescent Chemiluminescent in vivo Detection Detection HT PARP Pharmacodynamic Assay II HRP HRP # 4520-096-K Goat anti-rabbit IgG-HRP Goat anti-mouse IgG-HRP

Anti-PAR Polyclonal Anti-PAR Monoclonal

PAR PAR

Anti-PAR Monoclonal Immobilized Histones

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG PARP and PARG Assay Selection Guide

Summary of PARP and PARG Assay Details: y

Catalog # Application(s) Detection Method Test Sample Sensitivity Time* Page

Universal PARP Assay Kit** • Colorimetric Detection 4677-096-K PARP Inhibitor Screen Biotin NAD+ Histone ribosylation PARP Inhibitors 0.01-1 Units PARP 3 hr 9 • Chemiluminescence Detection 4676-096-K IC50 Determination PARP/Apoptosis Kit** • Colorimetric Detection 4684-096-K PARP Inhibitor Screen PAR Ab PARP Inhibitors 0.1 - 10 mUnits 3 hr • Chemiluminescence Detection 4685-096-K PARP Activity in Lysates Histone ribosylation Cell Lysates PARP 500 Cells 10

Homogeneous Fluorescence 4690-096-K HT PARP NAD+ Consumption PARP ribosylation PARP Inhibitors 10% PARP inhibition 1.5 hr 11 PARP Inhibition Assay Inhibitor Screen

PARP Inhibition in vivo PAR Ab PMBC Lysates PARP in vivo Pharmacodynamic Assay II** 4520-096-K 2-1000 pg/ml PAR 6.5 hr PAR Levels in Lysates In vivo PARylation Tissue Lysates 12 PARG Assay Kits** • Colorimetric Detection 4683-096-K Biotin NAD+ PARG Inhibitors 250 pg PARG 2 hr 14 • Chemiluminescence Detection 4682-096-K PARG Inhibitor Screen PAR degradation

*Actual Times may vary depending upon the number of samples being prepared and assayed. **Provided with a 96 strip well plate. PARP/ PAR/ PARG ASSAY SELECTION GUIDE ASSAY PARG PAR/ PARP/

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG Universal PARP Assay Kits

Poly (ADP-ribose) polymerase (PARP) catalyzes the NAD+-d ependent addition of poly (ADP-ribose) to itself and adjacent nuclear proteins, such as histones, in response to DNA damage. PARP contributes to the sequence of events that occurs during DNA base excision repair. Whereas PARP-mediated induction of necrosis can occur by extensive depletion of the intracellular NAD+ pool, the cleavage of PARP-1 promotes apoptosis by preventing DNA repair-induced survival and by blocking energy depletion-induced necrosis. Moreover, PARP inhibition promotes chemosensitization and radiosensitization of tumors. Trevigen's HT Universal PARP Assay Kits measure the incorporation of biotinylated poly(ADP-ribose) onto

histone proteins in a 96-well strip well format. This assay is ideal for the determination of IC50 values of known or suspected PARP inhibitors. UNIVERSAL PARP ASSAY For your convenience, Trevigen provides histone-coated strip wells. This format significantly reduces assay time.

FEATURES: OVERVIEW Trevigen’s • Colorimetric or Chemiluminescent readout Universal PARP Assay • 96 well format • Sensitive – detects 10 mU PARP/well Colorimetric or Chemiluminescent • Assay Time ~3 hrs Detection APPLICATIONS: HRP • Assay inhibitors and activators of PARP activity

Streptavidin • Determination of IC50 values for PARP Inhibitors

Biotin 009 KIT COMPONENTS:

PAR PARP-HSA Histone-Coated Strip Wells PARP Buffer and Cocktail Colorimetric or Chemiluminescent Detection Reagents Histones Activated DNA Strep HRP and Diluent 3-Aminobenzamide (known PARP inhibitor)

RELATED PRODUCTS: PARP Inhibitors (see page 23) ORDERING INFORMATION REFERENCES: 4667-50-06 Activated DNA for PARP Assay, 1. Satoh MS, Lindahl T. 1992. Role of poly(ADP-ribose) formation in DNA repair. 500 µl Nature 356: 356–8. 2. Golstein P, Kroemer G. 2007. Cell death by necrosis: Towards a molecular definition. 4676-096-K Trends Biochem Sci 32: 37-43. HT Universal Chemiluminescent PARP 3. D’Amours D, Sallmann FR, Dixit VM, Poirer GG. 2001. Gain-of-function of poly Assay Kit with Histone-coated Strip (ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for Wells, 96 tests apoptosis. J Cell Science 114: 3771-78. 4677-096-K 4. Eliasson M, Sampei K, Mandir AJ. 1997. Poly(ADP-ribose) polymerase gene disruption HT Universal Colorimetric PARP Assay renders mice resistant to cerebral ischemia. Nat Med 3:1089- 95. Kit with Histone-coated Strip Wells, 5. Jagtap P, Szabó C. 2005. Poly(ADP-ribose) polymerase and the therapeutic effects of its 96 tests inhibitors. Nat Rev Drug Discov. 4:421-40. 6. Pieper AA, Verma A, Zhang J, Snyder SH. 1999. Poly(ADP-ribose) polymerase, nitric 4677-096-P oxide and cell death. Trends Pharmacol Sci 20:171-81. Histone-Coated Natural Strip Wells, 7. Thiemermann C, Bowes J, Myint FP, Vane JR. 1997. Inhibition of the activity of 96 tests poly(ADP-ribose) synthase reduces ischemia-reperfusion injury in the heart and skeletal 4678-096-P muscle. Proc Natl Acad Sci USA 94:679-83. Histone-Coated White Strip Wells, 8. Kauppinen TM, Swanson RA. 2005. Poly(ADP-ribose) polymerase-1 promotes 96 tests microglial activation, proliferation, and matrix metalloproteinase-9-mediated neuron death. J Immunol. 174:2288-96. Bulk quantites available; please inquire 9. http://www.trevigen.com/MarketingFlyers/JDS_PARP2008_Poster.pdf

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG PARP/Apoptosis Assay Kits

During apoptosis, PARP-1 which catalyzes the NAD+-dependent addition of poly (ADP-ribose) (PAR) onto various cytoplasmic and nuclear proteins, is cleaved from about 116 kDa to 85 kDa. Moreover, this enzyme is a therapeutic target for BRCA1and BRCA2 associated breast cancers. The HT PARP/Apoptosis Assay is ideal for measuring the activity of PARP in cell extracts before and during apoptosis. This ELISA semi-quantitatively detects PAR deposited onto immobilized histone proteins in a 96-well format. An anti-PAR monoclonal antibody, goat anti-mouse IgG-HRP conjugate, and HRP substrate are used to generate a signal. Thus, signals correlate with PARP activity. Etoposide is a topoisomerase II inhibitor Trevigen’s PARP Apoptosis Assay that stabilizes this enzyme after it cleaves DNA. It is included as a control apoptosis inducer. Colorimetric or Chemiluminescent FEATURES: Detection • Colorimetric or Chemiluminescent readout

HRP • 96 well format • Highly sensitive – detects 0.1 mU PARP ~500 cells Goat anti-mouse IgG-HRP • Dynamic range between 0.1 to 10 mU PARP • Requires 10-100 ng extract for detection Anti-PAR Monoclonal • Assay Time ~3 hrs

PAR APPLICATIONS: • Measure activity in cells, primary or tumor cells • Measure activity before and after apoptosis

PARP/APOPTOSIS ASSAY PARP/APOPTOSIS Histones • Measure effect of PARP inhibitors using cell extracts

10 KIT COMPONENTS: ASSAY DESIGN: PARP-HSA and Buffer Anti-PAR Monoclonal Antibody Step 1: Ribosylation of histones by PARP. Activated DNA NAD+ • PAR polymer synthesized onto immobilized histones Histone-Coated Strip Wells PAR mAb and Diluent using PARP-HSA or cell extracts HRP Conjugate Etoposide • PARP inhibitors and caspase 3 cleavage prevent Colorimetric or Chemiluminescent Detection Reagents synthesis of PAR polymer. Step 2: Detection to measure incorporation of PAR attached to histones via PARP monoclonal antibody. REFERENCES: • Color/light output (Signal) is proportional to 1. Lawen A. 2003. Apoptosis—an introduction. BioEssays 25:888-896. PARP activity. 2. Okada H, Mak TW. 2004. Pathways of apoptotic and non-apoptotic death in tumor cells. • Monitor apoptosis by a decrease in PARP Signal. Nat Rev Cancer 4:592-603. 3. Miller MS, Zobre C, Lewis M. 1993. In vitro neuroprotective activity of inhibitors of poly-ADP ribose polymerase. Soc Neurosci Abstr 19.1656 4. Piper AA, Verma A, Zhang J, Snyder SH. 1999. Poly(ADP-ribose) polymerase, nitric oxide and cell death. Trends in Pharmacological Sciences 20:171-181. 5. Thiemermann C, Bowes J, Myint FP, and Vane JR. 1997. Inhibition of the activity of poly(ADP-ribose) synthase reduces ischemia-reperfusion injury in the heart and skeletal muscle. Proc Natl Acad Sci USA 94:679-683. 6. Virag L, Szabo C. 2002. The therapeutic potential of Poly(ADP-Ribose) Polymerase inhibitors. Pharmacological Reviews 54:375-429. 7. Baldwin EL, Osherhoff N. 2005. Etoposide, Topoisomerase II and Cancer. Curr. Med. Chem. Anti-Cancer Agents 5:363-372. 8. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, Kyle S, Meuth M, Curtin NJ, Helleday T. 2005. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434:913-7. 9. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, Santarosa M, ORDERING INFORMATION Dillon KJ, Hickson I, Knights C, Martin NM, Jackson SP, Smith GC, Ashworth A. 2005. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 4684-096-K HT Universal Colorimetric 434:917-21. PARP/Apoptosis Assay Kit, 96 tests 10. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, Mortimer P, Swaisland H, Lau A, O'Connor MJ, Ashworth A, Carmichael J, Kaye SB, Schellens JH, de Bono JS. 2009. 4685-096-K Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. HT Universal Chemiluminescent N Engl J Med 361:123-34. PARP/Apoptosis Assay Kit, 96 tests

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG HT Fluorescent Homogeneous PARP Inhibition Assay

Trevigen's HT Fluorescent Homogeneous PARP Inhibition Assay is a highly sensitive fluorescent screening assay for the rapid identification of PARP-1 inhibitors in an in vitro system. This one hour endpoint assay is performed in two successive steps requiring only the consecutive addition of reaction components. A PARP autoribosylation reaction is first performed, followed by a detection step. The remaining NAD+ level is determined in a cycling reaction involving alcohol dehydrogenase and diaphorase. Each time NAD+ cycles through these coupled reactions, a molecule of highly fluorescent resorufin is generated.

The assay can be used to determine relative IC50 values for PARP inhibitors and is capable of detecting as little as 10% inhibition of PARP-1 activity. Inhibitors are identified by an increase in fluorescent signal when PARP mediated NAD+ depletion is inhibited. PARP INHIBITION ASSAY FEATURES: • Highly sensitive fluorescent assay • Detect as little as 10% inhibition of PARP Activity • Inhibitors identified by increase in Fluorescent Signal OVERVIEW • No wash steps • 96 test size • Rapid – Assay takes ~ 1.5 hr

APPLICATIONS: • Ideal for screening compounds which potentially inhibit PARP activity

• Determination of relative IC50 values for PARP Inhibitors KIT COMPONENTS: 0011 PARP-HSA and Buffer Activated DNA NAD+ 10X Cycling Enzymes 10X Resazurin Stop Solution

REFERENCES: 1. Meyer-Ficca, M.L., et al., 2005. Poly(ADP-ribose) polymerases: managing genome stability. Int J Biochem Cell Biol, 37:920-6. 2. Hassa, P.O., et al., 2006. Nuclear ADP-ribosylation reactions in mammalian cells: where are we today and where are we going? Microbiol Mol Biol Rev, 70:789-829. 3. Virag, L. and C. Szabo, 2002. The therapeutic potential of poly(ADP-ribose) polymerase inhibitors. Pharmacol Rev 54:375-429. 4. Tutt, A.N., et al., 2005. Exploiting the DNA repair defect in BRCA mutant cells in the design of new therapeutic strategies for cancer. Cold Spring Harb Symp Quant Biol, 70:139-48. 5. Bryant, H.E., et al., 2005. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434:913-7. 6. Ratnam, K. and J.A. Low 2007. Current development of clinical inhibitors of poly(ADP-ribose) polymerase in oncology. Clin Cancer Res 13:1383-8.

PARP Homogeneous Assay ASSAY DESIGN: Step 1: PARP reaction requiring NAD+ is performed. poly-ADP ribose Step 2: Cycling Assay • As PAR polymer is synthesized NAD+ is consumed PARP 1 AD from the reaction mixture. inactive EtOH Acetaldehyde • PARP inhibitors prevent NAD+ consumption. Step 2: Cycling Assay is performed to quantify nicotinamide [NAD+] NADH +H ORDERING INFORMATION NAD+ remaining from Step 1. • Each time NAD+ cycles, a molecule of highly 4690-096-K Activated + PARP 1 fluorescent resorufin is generated. DNA active HT Fluorescent Homogeneous PARP Diaphorase • PARP inhibitors are identified by an increase Inhibition Assay, 96 tests Step 1: PARP Reaction in fluorescent Signal. Fluorescent Resorufin Resazurin hv ~ [NAD+] remaining after Step 1 1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:11 PM Page 16

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PARP/ PAR/ PARG HT PARP in vivo Pharmacodynamic Assay II

PARP catalyzes the NAD+-dependent addition of poly (ADP-ribose) (PAR) onto itself and adjacent nuclear proteins. This enzyme is a therapeutic target for BRCA1 and BRCA2 associated breast cancers. To address the need to monitor PARP activity among different individuals and within cells, Trevigen's improved and validated HT PARP in vivo Pharmacody- Trevigen’s PARP namic Assay II measures net PAR levels in tissue or cellular extracts and has been used to Pharmacodynamic Assay document differences in PAR levels among tumor lysates, organs and xenografts. The HT

Chemiluminescent PARP in vivo Pharmacodynamic Assay II employs a 96 well plate, pre-coated with Trevigen’s Detection monoclonal PAR antibody as the capture agent, and anti-PAR polyclonal rabbit antibody as the detecting agent. HRP FEATURES: Goat anti-rabbit IgG-HRP • Pre-coated 96 well capture antibody plates • High signal to noise ratio Anti-PAR Polyclonal • Detection sensitivity of 2 pg/ml of PAR • Broad linear dynamic range to 1,000 pg/ml PHARMACODYNAMIC ASSAY PHARMACODYNAMIC ASSAY PAR • Reduced inter-assay variability • Validated assay that measures drug action on PARP in both in vivo and in vitro settings Anti-PAR Monoclonal • 96 test size Immobilized IN VIVO APPLICATIONS: • Quantitation of PAR in peripheral blood mononuclear cells, tissue culture cells, and tumor

PARP PARP lysates from different tissues, organs and xenografts. ASSAY DESIGN: • Monitoring the efficacy of PARP inhibitors on PAR formation in vivo. Step 1: Immobilized PAR mAb captures cellular PAR • Verifying observations of enhanced cancer cell cytotoxicity arising from PARP inhibitor/ 12 and PAR attached to proteins in prepared lysates. anticancer drug combination therapy. Step 2: Binding of PAR polyclonal detecting Ab to captured PAR. • Facilitating development of PARP and PARG targeted therapeutics. Step 3: Measure captured PAR via binding of goat anti-rabbit IgG-HRP with chemiluminescent detection. KIT COMPONENTS: • Light output (Signal) correlates with the amount of PAR Standard and Sample Buffer PAR Polyclonal Detecting Ab and Diluent cellular PAR. Goat anti-Rabbit IgG-HRP Detection Reagents • Allows quantification of PAR in biopsies of tumor Cell Lysis Reagent and 20% SDS DNase I and Mg Cation xenografts, tissue samples, peripheral blood Jurkat Cell Lysate Standard Controls mononuclear cells and tissue culture cells. (Low, Medium, and High) Pre-coated 96-stripwell plate and sealers

REFERENCES: 1. Virag, L., Szabo, C. 2002. The therapeutic potential of Poly(ADP-Ribose) Polymerase inhibitors. Pharmacological Reviews 54:375-429. 2. Curtin NJ. 2005. PARP inhibitors for cancer therapy. Expert Rev Mol Med 7:1-20. 3. Kinders JK, Hollingshead M, Khin S, Rubinstein L, Tomaszewski JE, Doroshow JH, Parchment RE, and the National Cancer Institute Phase 0 Clinical Trials Team. 2008. Preclinical Modeling of a Phase 0 Clinical Trial: Qualification of a Pharmacodynamic Assay of Poly (ADP-Ribose) Polymerase in Tumor Biopsies of Mouse Xenografts. Clin Cancer Res 14:6877-85. 4. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, Kyle S, Meuth M, Curtin NJ, Helleday T. 2005. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434:913-7. 5. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, Santarosa M, Dillon KJ, Hickson I, Knights C, Martin NM, Jackson SP, Smith GC, Ashworth A. 2005. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434:917-21. ORDERING INFORMATION 6. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, Mortimer P, Swaisland H, Lau A, O'Connor MJ, Ashworth A, Carmichael J, Kaye SB, Schellens JH, de Bono JS. 2009. 4520-096-K Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. HT PARP in vivo Pharmacodynamic Assay II, 96 tests N Engl J Med 361:123-34.

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG HT PARP in vivo Pharmacodynamic Assay II

ASSAY VALIDATION:

PAR level measured in Jurkat, PBMC and Xenograft cell lysate PARP

1000.0 IN VIVO

800.0

600.0 PHARMACODYNAMIC ASSAY

400.0 PAR (pg/ml) PAR PAR (pg/ml)

200.0

0.0 OVERVIEW Jurkat (High) Jurkat Jurkat (Low) PBMC Xeno (Medium)

Assay Validation Using Control Lysates

PAR levels in three donors' PBMC lysates

120.0

100.0 0013 80.0

60.0 PAR (pg/ml) PAR

PAR (pg/ml) PAR 40.0

20.0

0.0 J1 J2 J3 K1 K2 K3 L1 L2 L3

Assay Validation Using Donor Cells. Cells obtained from three different donors were lysed and assayed on three different days.The means and standard deviations of each determination are shown.

PAR Standard Curve

2000000

1500000

1000000 y = 1778.6x + 34596 Net Mean RLU R2 = 0.9945 Net Mean RLU Mean Net 500000

0 0 200 400 600 800 1000 1200 pg /ml PAR

Assay Validation Standard Curve: Typical PAR standard curve with trend line. The linear dynamic range is from 10 to 1000 pg/ml.The R2 value for the linear curve fit (shown) was 0.9945.

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG HT Universal PARG Assay Kits

Poly(ADP-ribose) glycohydrolase (PARG) degrades polymers synthesized by poly Trevigen’s (ADP-ribose) polymerase (PARP1). When activated by DNA strand breaks, PARP1 uses Universal PARG Assay NAD+ as a substrate to form ADP-ribose polymers on itself and on specific acceptor proteins. These polymers are in turn rapidly degraded by PARG, a ubiquitously expressed exo- and Colorimetric or endoglycohydrolase. PARG may maintain the active state of PARP1 by continuously Chemiluminescent Detection removing inhibitory ADP-ribose residues from PARP1. The regulation of PARG activity may therefore influence PARP-mediated cell death. PARG inhibitors could thereby indirectly HRP inhibit PARP1 activity. Trevigen’s HT PARG Assay Kits measure the loss of biotinylated PAR from histones attached to strip wells in a 96 well format and is ideal for the screening of Streptavidin PARG inhibitors and for measuring the activity of PARG in cell extracts. Biotin FEATURES: PAR • Colorimetric or Chemiluminescent readout • 96 well format • Highly sensitive – detects 50 pg PARG per well • Assay Time ~2 hrs Histones APPLICATIONS:

ASSAY DESIGN: • Measure PARG Activity in cell extracts Step 1: Ribosylation of histones by PARP. • Identify inhibitors and activators of PARG activity • Biotinylated-PAR polymer synthesized onto • Determination of IC50 values for PARG Inhibitors

PARG ASSAY ASSAY PARG immobilized histones using PARP-HSA. Step 2: PARG degrades PAR. KIT COMPONENTS: 14 • Biotinylated-PAR attached to histones is hydrolyzed PARP-HSA and PARG Histone-Coated Strip Wells by PARG. Buffers and Cocktail Activated DNA Step 3: Detection to measure remaining biotinylated PAR attached to histones via Strep-HRP. Strep HRP and Diluent DEA (known PARG inhibitor) • PARG inhibitors are identified by an increase in Signal. Colorimetric or Chemiluminescent Detection Reagents

REFERENCES: 1. Koh DW, Dawson VL, Dawson TM. 2005. The road to survival goes through PARG. Cell Cycle. 4:397-399. 2. Cuzzocrea S, Wang ZQ. 2005. Role of poly(ADP-ribose) glycohydrolase (PARG) in shock, ischemia and reperfusion. Pharmacol Res. 52:100-108. 3. Bonicalzi ME, Haince JF, Droit A, Poirier GG. 2005. Regulation of poly(ADP-ribose)metabolism by poly(ADP-ribose) glycohydrolase: where and when? Cell Mol Life Sci. 62:739-750. 4. Patel NS, Cortes U, Di Poala R, Mazzon E, Mota-Filipe H, Cuzzocrea S, Wang ZQ, Thiemermann C. 2005. Mice lacking the 110-kD isoform of poly(ADP-ribose) glycohydrolase are protected against renal ischemia/reperfusion injury. J Am Soc Nephrol. 16:712-719. 5. Patel CN, Koh DW, Jacobson MK, Oliveira MA. 2005. Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain. Biochem J. 388:493-500. 6. Falsig J, Christiansen SH, Feuerhahn S, Burkle A, Oei SL, Keil C, Leist M. 2004. Poly(ADP-ribose) glycohydrolase as a target for neuroprotective intervention: assessment of currently available pharmacological tools. Eur J Pharmacol. 497:7-16. 7. Cortes U, Tong WM, Coyle DL, Meyer-Ficca ML, Meyer RG, Petrilli V, Herceg Z, Jacobson EL, Jacobson MK, Wang ZQ. 2004. Depletion of the 110-kilodalton isoform of poly(ADP-ribose) glycohydrolase increases sensitivity to genotoxic and endotoxic stress in mice. Mol Cell Biol. 24:7163-7178. 8. Hanai S, Kanai M, Ohashi S, Okamoto K, Yamada M, Takahashi H, Miwa M. 2004. Loss of ORDERING INFORMATION poly(ADP-ribose) glycohydrolase causes progressive neurodegeneration in Drosophila melanogaster. Proc Natl Acad Sci U S A. 101:82-86. 4683-096-K HT Universal Colorimetric PARG Assay 9. Lu XC, Massuda E, Lin Q, Li W, Li JH, Zhang J. 2003. Post-treatment with a novel PARG Kit, 96 tests inhibitor reduces infarct in cerebral ischemia in the rat. Brain Res. 18:99-103. 10. Ying W, and Swanson RA. 2000. The poly(ADP-ribose) glycohydrolase inhibitor 4682-096-K gallotannin blocks oxidative astrocyte death. Neuroreport. 11:1385-1388. HT Universal Chemiluminescent PARG Assay Kit, 96 tests

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG PARP

ADP-Ribose Polymerase

Trevigen offers two grades of poly (ADP-ribose) polymerase: “standard” PARP, and high specific activity (HSA) enzyme. The specific activity serves as a measure of enzyme purity, with the standard enzyme being purified to approximately 50% purity, and the HSA grade to greater than 95% purity. The standard PARP enzyme is useful as a positive control for both Western blot analysis and analysis of protein ribosylation. For more detailed studies or for drug discovery applications, PARP HSA is recommended. Enzyme preparations with lower specific activity are available (catalog #s 4667-50-EB and 4667-250-EB). High specific activity PARP (PARP-HSA) is used in Trevigen’s PARP Assay Kits. PARP is activated by 10X Activated DNA(catalog # 4671-096-06), which can be assayed using Trevigen’s Universal PARP Activation kits (catalog #s 4677-096-K; 4676-096-K see page 9), or PARP Apoptosis Assay kits (catalog #s 4684-096-K; 4685-096-K see page 10).

SOURCE: OVERVIEW Purified from E. coli containing a recombinant plasmid harboring the human PARP gene.

UNIT DEFINITION: One unit of PARP incorporates 10 femtomoles of NAD+ onto immobilized histones in 30 minutes. High specific activity PARP (PARP-HSA) is provided at 10 units/µl. For enzyme preparations with PARP lower specific activity, (catalog #s 4667-50-EB and 4667-250-EB), the number of units provided (>5 Units/µl) will vary.

APPLICATIONS: 0015 Identification of inhibitors and activators of PARP activity Quantitation of DNA Damage Investigation of PARP inactivation during apoptosis Western Blot Analysis

STORAGE BUFFER: 20 mM Tris-Cl (pH 8.0), 200 mM NaCl, 1 mM DTT, 0.1% Triton X-100, 50% glycerol, and 0.1 mg/ml BSA.

STORAGE: Store at -20°C in a manual defrost freezer. For long-term storage, freeze in working aliquots at -80°C. Avoid freeze-thaw cycles.

ORDERING INFORMATION REFERENCES: 1. Satoh, M.S. and T. Lindahl. 1994. Role of poly(ADP-ribose) formation in DNA repair. 4668-100-01 Nature 356:356-358. PARP Enzyme, High Specific Activity, 2. Lautier, D., J. Lagueux, J. Thibodeau, L. Menard, and G.G. Poirier. 1993. Molecular and 1,000 units biochemical features of poly(ADP-ribose) metabolism. Mol Cell Biochem 122:171-193. 4668-500-01 3. Curtin NJ. 2005. PARP inhibitors for cancer therapy. Expert Rev Mol Med. 7:1-20. + PARP Enzyme, High Specific Activity, 4. Kim MY, Mauro S, Gevry N, Lis JT, Kraus WL. 2004. NAD -Dependent Modulation of 5,000 units Chromatin Structure and Transcription by Nucleosome Binding Properties of PARP-1. Cell 119:803–814. 4668-02K-01 5. Virag, L., and Szabo, C. 2002. The therapeutic potential of Poly(ADP-Ribose) Polymerase PARP Enzyme, High Specific Activity, inhibitors. Pharmacological Reviews 54:375-429. 20,000 units 6. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, Kyle S, Meuth M, Curtin NJ, Helleday T. 2005. Specific killing of BRCA2-deficient tumours with inhibitors of poly 4667-50-EB (ADP-ribose) polymerase. Nature 434:913-917. Human PARP Enzyme and Buffer, 50 µl 7. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, Santarosa M, Dillon 4667-250-EB KJ, Hickson I, Knights C, Martin NM, Jackson SP, Smith GC, Ashworth A. 2005. Targeting the Human PARP Enzyme and Buffer, 250 µl DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434:917-921. 8. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, Mortimer P, Swaisland H, 4671-096-06 Lau A, O'Connor MJ, Ashworth A, Carmichael J, Kaye SB, Schellens JH, de Bono JS. 2009. 10X Activated DNA, 300 µl Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med 361:123-134. 1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:12 PM Page 20

DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG PARG

Poly(ADP-ribose) glycohydrolase

Poly(ADP-ribose) glycohydrolase (PARG) degrades poly(ADP-ribose) (PAR) polymers synthesized by poly(ADP-ribose) polymerase (PARP-1). PARG is ideal for use as a positive control in Trevigen’s PARG Assay Kit (catalog # 4682-096-K and 4683-096-K see page 14) and for Western blot analysis of PARG in cell extracts.

SOURCE: Purified from E. coli containing a recombinant plasmid harboring the catalytic domain of the bovine PARG gene.

MEASUREMENT OF ACTIVITY: PARG activity is measured by the loss of biotinylated PAR from immobilized histones in 30 minutes.

APPLICATIONS: • Identification of inhibitors and activators of PARG activity • Western Blot Analysis

STORAGE BUFFER: ® 20 mM KPO4 (pH 7.2), 50 mM KCl, 0.1 mg/ml BSA, 0.1% Triton X-100, 1mM DTT,

PARG 50% Glycerol.

16 STORAGE: Stable for at least one year when stored at −20 °C.

REFERENCES: 1. Koh DW, Dawson VL, Dawson TM. 2005. The road to survival goes through PARG. Cell Cycle 4:397-399. 2. Oei SL, Keil C, Ziegler M. 2005. Poly(ADP-ribosylation) and genomic stability. Biochem Cell Biol 83:263-269. 3. Cuzzocrea S, Wang ZQ. 2005. Role of poly(ADP-ribose) glycohydrolase (PARG) in shock, ischemia and reperfusion. Pharmacol Res. 52:100-108. 4. Bonicalzi ME, Haince JF, Droit A, Poirier GG. 2005. Regulation of poly(ADP-ribose) metabolism by poly(ADP-ribose) glycohydrolase: where and when? Cell Mol Life Sci. 62:739-750. 5. Patel NS, Cortes U, Di Poala R, Mazzon E, Mota-Filipe H, Cuzzocrea S, Wang ZQ, Thiemermann C. 2005. Mice lacking the 110-kD isoform of poly(ADP-ribose) glycohydrolase are protected against renal ischemia/reperfusion injury. J Am Soc Nephrol. 16:712-719. 6. Patel CN, Koh DW, Jacobson MK, Oliveira MA. 2005. Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain. Biochem J. 388:493-500.

ORDERING INFORMATION

4680-096-01 PARG Enzyme (1 µg/ml), 300 µl

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG PAR

Poly(ADP-ribose) Polymer

PAR was synthesized using poly(ADP-ribose) polymerase (PARP) in the presence of NAD+, cleaved from PARP, and subsequently purified. The PAR chain length ranges from 2 to 300 monomers, and it is recognized by Trevigen’s anti-PAR monoclonal (catalog # 4335-MC-100 see page 19) and polyclonal (catalog # 4336-BPC-100 see page 20) antibodies.

APPLICATIONS: Immunodetection as a positive control in ELISA and Western Blot Analysis.

STORAGE: PAR is provided in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA and stored at -80°C. It may be aliquoted to avoid repeated freeze-thawing.

REFERENCES: OVERVIEW 1. Shah, G.M., et al. 1995. Methods for biochemical study of poly(ADP-ribose) metabolism in vitro and in vivo. Anal Biochem 227:1-13. 2. Affar, E.B., et al. 1998. Immunodot blot method for the detection of poly(ADP-ribose) synthesized in vitro and in vivo. Anal Biochem 259:280-283.

3. Menard, L. and G.G. Poirier. 1987. Rapid assay of poly (ADP-ribose) glycohydrolase. PAR Biochem Cell Biol 65:668-673.

0017

ORDERING INFORMATION 4336-100-01 Poly (ADP) Ribose (PAR) Polymer, 100 µl

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG Anti-PARP Monoclonal Antibody C2-10

The anti-poly (ADP-ribose) polymerase (PARP) mouse monoclonal antibody may be used for the analysis of PARP protein levels and the detection of cleavage of PARP in apoptotic cells. During apoptosis, PARP-1 activity initially increases, but later falls due to auto-modification and cleavage by caspases. The specific rate of change in PARP-1 activity is dependent upon Wehi Jurkat CCRF-CEM a variety of factors including cell type, method of induction of DNA damage or apoptosis, and culture conditions. Specific proteolytic cleavage of PARP has been demonstrated to be a reliable marker for apoptosis in a wide variety of cell types, generating 85 kDa and 26 kDa fragments.

PHYSICAL STATE:

C2-10 is an IgG1 and is provided in 1X PBS/0.1 mg/ml BSA/0.01% sodium azide.

IMMUNOGEN: Calf Thymus PARP-1

SPECIFICITY: C2-10 detects the 114 kDa PARP-1 holoenzyme, 85 kDa apoptosis-related, and necrosis-related 50/ 62/74 kDa cleavage fragments. The antibody cross-reacts with human, monkey, hamster, rat and mouse PARP-1 but not chicken.

APPLICATIONS:

ANTI-PARP MONOCLONAL ANTIBODY C2-10 ANTI-PARP Western Blot Analysis and Immunocytochemistry. For Western blotting, a 1:2000 dilution using enhanced chemiluminescence (ECL) or 1:1000 dilution using alkaline phosphatase is Western blot analysis of Wehi, Jurkat and CCRF-CEM cell recommended. 18 lines untreated (H) and treated (T) with 25 µM Etoposide for 16 hours at 37oC. Cells were lysed in Tris-Glycine SDS sample buffer at the concentration 1 x 107 cells/ml and CONTROL: 10 µl of each lysate were loaded per well of a 4-20% PARP HSA Enzyme (Catalog # 4668-100-01) available separately. Tris-Glycine gel. Proteins were transferred onto an Immobilon FL membrane and PARP was detected with STORAGE: Trevigen’s anti-PARP (C2-10) antibody followed by an IR800-conjugated secondary antibody. The membrane was Store at 4°C for at least 1 month, or store aliquots at -20°C. scanned using an Odyssey Infrared Imaging System (Licor). REFERENCES: 1. Lamarre D, B. Talbot, G de Murcia, C Laplante, Y Leduc, A Mazen, GG Poirier. 1988. Structural and functional analysis of poly(ADP-ribose) polymerase: an immunological study. Biochim. Biophys. Acta 950:147-60. 2. Simonin F, JP Briand, S Muller, G de Murcia 1991 Detection of Poly(ADP-ribose) Polymerase in Crude Extracts by Activity-Blot. Anal Biochem. 195:226-31. 3. Shah GM, D Poirier, C Duchaine, G Brochu, S Desnoyers, J Lagueux, A Verreault, JC Hoflack, JB Kirkland, GG Poirier. 1995 Methods for biochemical study of poly(ADP-ribose) metabolism in vitro and in vivo. Anal Biochem. 227:1-13.

ORDERING INFORMATION

4338-MC-50 Anti-PARP Monoclonal Antibody (clone C2-10), 50 µg 4668-100-01 PARP Enzyme, High Specific Activity, 1000 Units

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG Anti-PAR Monoclonal Antibody

CONTENTS: Catalog # Description Size 4335-MC-100 Anti-PAR monoclonal 100 µl 4500-10-P PARP treated protein control 10 µl ANTI-PAR MONOCLONAL ANTIBODY This monoclonal antibody is specific for poly(ADP-ribose) (PAR) polymer. It can be used to detect ribosylated proteins by ELISA, Western blot, immunocytochemistry in situ, and for immunopurification. It is supplied with a positive control for ELISA and Western blot analysis Western blot analysis of Wehi and Jurkat cells using consisting of poly-ADP-ribosylated PARP-1 protein (PARP-PAR). Trevigen’s anti-PAR monoclonal antibody. Cells were lysed in Tris-Glycine SDS sample buffer at the concentration 1x PHYSICAL STATE: 7 10 cells/ml and 10 µl of each lysate were loaded per well This antibody is an IgG and is provided as purified immunoglobulin from mouse ascites in on 4-20% Tris-Glycine gel. Proteins were transferred onto 3a an Immobilon FL membrane and ribosylated proteins were 1X PBS containing 50% glycerol at 1 mg/ml. PARP-PAR is provided at 75 µM in 10 mM

detected with Trevigen’s anti-PAR antibody (catalog # 4335- Tris-HCl (pH 8.0), 1 mM EDTA. OVERVIEW MC-100) followed by IR800-conjugated secondary antibody (Licor). For an absorption control (right panel), the anti-PAR IMMUNOGEN: antibody was pre-incubated with PAR polymer (#4336-100- 01). Membranes were scanned using an Odyssey Infrared Purified ADP-ribose polymers between 2 and 50 units long Imaging System (Licor). SPECIFICITY: The antibody is specific for PAR polymers 2 to 50 units long, but does not recognize + 200> structurally related RNA, DNA, ADP-ribose monomers, NAD , or other nucleic acid monomers.

APPLICATIONS: 0019 ELISA, Western Blot Analysis, immunoprecipitation, and immunopurification. For Western blots, an antibody dilution of 1:1000 is recommended.

STORAGE: Store the anti-PAR monoclonal at -20°C (manual defrost freezer). Store the PARP-treated protein control at -80°C.

Western blot analysis of parylated PARP (catalog # 4500- REFERENCES: 10-P). 15 µl of a 1:10 dilution of PARP-PAR in sample buffer were separated on a 4-20% Tris-Glycine SDS-PAGE gel and 1. Kupper, J.H., G. de Murcia, and A. Burkle. 1990. Inhibition of poly(ADP-ribosyl)ation by blotted on to PVDF membrane. overexpressing the poly(ADP-ribose) polymerase DNA binding domain in mammalian cells. J Biol Chem 265:18721-18724. Ribosylated proteins were detected with 1:1000 dilution 2. Malik, N., M. Miwa, T. Sugimura, P. Thraves, and M. Smulson. 1983. Immunoaffinity of anti-PAR followed by a 1:5000 dilution of HRP fractionation of the poly(ADP-ribosyl)ated domains of chromatin. PNAS USA 80:2554-2558. conjugated goat anti-mouse IgG and visualized using chemiluminescence. 3. Kawamitsu, H., H. Hoshino, H. Okada, M. Miwa, H. Momoi, and T. Sugimura. 1984. Monoclonal antibodies to poly(adenosine diphosphate ribose) recognize different structures. ORDERING INFORMATION Biochem 23:3771- 3777. 4. Affar, E.B., et al. 1998. Immunodot blot method for the detection of poly(ADP-ribose) 4335-AMC-050 synthesized in vitro and in vivo. Anal Biochem 259:280-283. Anti-PAR Monoclonal Affinity Purified, 5. Ismail, I.H., S. Nystrom, J. Nygren, and O. Hammarsten. 2005. Activation of ataxia 50 µl telangiectasia mutated by DNA strand break-inducing agents correlates closely with the 4335-MC-100 number of DNA double strand breaks. J Biol Chem 280:4649-4655. Anti-PAR Monoclonal Antibody, 100 µl 6. Gagné, J-P., M. Isabelle, K.S. Lo, S. Bourassa, M.J. Hendzel, V.L. Dawson, T.M. Dawson, and G.G. Poirier. 2008. Proteome-wide identification of poly (ADP-ribose) binding proteins 4335-MC-100-AC and poly(ADP-ribose)-associated protein complexes. Nucleic Acids Res 36:6959-6976. Anti-PAR Monoclonal Antibody, 100 µl, and PARP-PAR control protein, 10 µl

4335-MC-01K-AC Anti-PAR Monoclonal Antibody, 1000 µl, and PARP-PAR control protein, 100 µl 4500-10-P PARP treated protein control, 10 µl

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG Anti-PAR Rabbit Polyclonal Antibody

A rabbit polyclonal antibody raised against poly(ADP-ribose) (PAR) polymer. The anti-PAR polyclonal antibody can be used to detect ribosylated proteins by immunodetection. Trevigen’s PARP treated control protein (4500-10-P see page 19) and PAR polymer (catalog # 4336-100-01 see page 17) may be used as positive controls.

PHYSICAL STATE: This polyclonal antibody is a purified IgG fraction in 1X PBS containing 50% glycerol.

IMMUNOGEN: Poly(ADP-ribose) polymer

SPECIFICITY: This polyclonal antibody detects free PAR and poly-ribosylated proteins.

APPLICATIONS: For Western and dot blotting, an antibody dilution of 1:1000 is recommended. For ELISA a 1:4000 antibody dilution is recommended. Empirical determination of antibody dilutions will be required for optimum results.

STORAGE: -20°C (manual defrost freezer) ANTI-PAR RABBIT POLYCLONAL ANTIBODY RABBIT POLYCLONAL ANTI-PAR REFERENCES: 20 Western blot analysis of Healthy and Etoposide treated Wehi 1. Affar, E.B., et al. 1998. Immunodot blot method for the detection of poly (ADP-ribose) cells using Trevigen’s anti-PAR polyclonal antibody. synthesized in vitro and in vivo. Anal. Biochem. 259:280-283. 2. Shah, G.M., et al. 1995. Methods for biochemical study of poly (ADP-ribose) metabolism in vitro and in vivo. Anal. Biochem. 227:1-13. 3. Kinders JK, Hollingshead M, Khin S, Rubinstein L, Tomaszewski JE, Doroshow JH, Parchment RE, and the National Cancer Institute Phase 0 Clinical Trials Team. 2008. Preclinical Modeling of a Phase 0 Clinical Trial: Qualification of a Pharmacodynamic Assay of Poly (ADP-Ribose) Polymerase in Tumor Biopsies of Mouse Xenografts. Clin Cancer Res 14:6877-85.

ORDERING INFORMATION

4336-APC-050 Anti-PAR Polyclonal Affinity Purified, 50 µl 4336-BPC-100 Anti-PAR Polyclonal Antibody, 100 µl

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG Biotin-NAD+

6-Biotin-17-NAD+

Biotinylated NAD+ provides a convenient non-isotopic alternative to radiolabeled NAD+ for determination of IC50 values for candidate PARP inhibitors and studies requiring this substrate. A number of proteins, including poly (ADP-ribose) polymerase (PARP), use NAD+ as a substrate for their function. The PARP enzyme catalyzes the polymerization of ADP-ribose onto a number of nuclear proteins. Biotinylated NAD+ allows an indirect measure of PARP activity when biotin incorporation is detected using a conjugated-streptavidin detection system.

CONCENTRATION: 250 µM in water

MOLECULAR WEIGHT:

1180.08 g/mole (1158.09 g/mole as a free acid) OVERVIEW BIOTIN-NAD EXTINCTION COEFFICIENT: 22,000 at 265 nm

PURITY: Greater than 95% by HPLC + STORAGE: Avoid repeated freeze thaw cycles, prepare aliquots and store at -80°C. 0021 REFERENCES: 1. Zhang J, Snyder SH. 1993. Purification of a nitric oxide-stimulated ADP-ribosylated protein using biotinylated beta-nicotinamide adenine dinucleotide. Biochemistry 32:2228-33. 2. http://www.trevigen.com/MarketingFlyers/JDS_PARP2008_Poster.pdf

S H N N O H O NH o

HN NH O HN NH 2 N O N O O + N OOP O P N N - - O OH HO O O O

OH OH ORDERING INFORMATION Na+

4670-500-01 Biotin-NAD+, 500 µl

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG Fluorescein-NAD+

Fluorescein-NAD+ (6-fluorescein-17-nicotinamide-adenine-dinucleotide)

Fluorescein-NAD+ provides a convenient, non-isotopic alternative to radiolabeled NAD for use with enzymes requiring NAD+ as a substrate or cofactor. A number of proteins, including poly (ADP-ribose) polymerases (e.g. PARP-1, PARP-2), and the SIR2 family of NAD+-dependent histone/protein deacetylases, use NAD+ as a substrate for their function. Fluorescein-conjugated NAD+ permits the direct measurement of PARP and other NAD+-dependent enzymes by fluorescence microscopy. Fluorescein-NAD+ enters cells with the aid of Trevigen’s Cell Permeabilization Solution (catalog # 4674-250-01).

PHYSICAL STATE: Provided in solution at a concentration of 250 µM in deionized water. There is a 1:1 stoichiometry for incorporation; one Fluorescein label for each NAD+ molecule. +

EXTINCTION COEFFICIENT: 38,000 at 262 nm

STORAGE: Fluorescent Image of In-Cell Assay Store at - 80°C Incorporation of Fluorescein-labeled ribose from Fluorescein-NAD+ by Poly (ADP-ribose) polymerase (PARP) APPLICATIONS: in presence of Evan’s Blue. FLUORESCEIN-NAD • Activity measurements of NAD+-requiring enzymes. • Assays to identify inhibitors of activators of NAD+-requiring enzymes. 22 *Cell Permeabilization Solution (catalog # 4674-250-01), is required for in-cell assays for Fluorescein-NAD+ entry.

REFERENCES:

H 2 0 2 3-AB 1. Bakondi E, Bai P, Szabó E E, Hunyadi J, Gergely P, Szabó C, Virág L. (2002) Detection of poly(ADP-ribose) polymerase activation in oxidatively stressed cells and tissues using biotinylated NAD+ substrate. J Histochem Cytochem 50:91-8. Light

Fluorescein Cell Permeabilization Solution

Cell Permeabilization Solution allows for the efficient transfer of Fluorescein-conjugated Merge NAD+ across cellular membranes. The solution contains a selective cell permeable peptide + to deliver Trevigen’s Fluorescein-NAD (catalog # 4673-500-01) from the outside to the inside of intact cells.

Wehi cells treated with hydrogen peroxide or 3-AB (PARP inhibitor) PHYSICAL STATE: + were incubated in a PARP cocktail containing Fluorescein-NAD and Peptide provided as solution in deionized water. Cell Permeabilization Solution were visualized by light and fluorescent microscopy. STORAGE: Store at -20°C

APPLICATIONS: • In-cell activity measurements of NAD+-requiring enzymes. • In-cell Assays to identify inhibitors of activators of NAD+-requiring enzymes.

REFERENCES: ORDERING INFORMATION 1. Fischer, R., Fotin-Mleczek, M., Hufnagel, H. and Brock, R. (2005), Break on through to the 4673-500-01 Other Side—Biophysics and Cell Biology Shed Light on Cell-Penetrating Peptides. Fluorescein-NAD+, 250 µl ChemBioChem, 6: 2126–2142. 4674-250-01 Cell Permeabilization Solution, 250 µl

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DNA DAMAGE AND GENOMIC INSTABILITY

PARP/ PAR/ PARG PARP and PARG Inhibitors

3-Aminobenzamide Catalog # 4667-50-03 3-Aminobenzamide is a poly(ADP-ribose) polymerase (PARP) inhibitor in the family of benzamides. 3-aminobenzamide attenuates tissue injury following transient ischemia and acts as a neuroprotectant since it inhibits PARP, an enzyme activated by peroxynitrite activity. This PARP inhibitor, with low in vivo toxicity, has been analyzed in the range of 0.1 µ

to 10,000 M, and is provided at 200 mM in ethanol for subsequent serial dilution and use PARRP AND PARG INHIBITORS with Trevigen’s HT Universal PARP Assay Kits.

4-Amino-1,8-naphthalimide Catalog # 4667-50-09 4-amino-1,8-naphthalimide is a potent inhibitor of poly (ADP-ribose) polymerase (PARP)

and also reduces ischemia-reperfusion injury in the heart and skeletal muscle. It is OVERVIEW 1000-fold more potent that 3-aminobenzamide and exhibits mixed-type inhibition with respect to the substrate NAD+, at micromolar concentrations. This inhibitor, which has been analyzed at a concentration of 20 µM, is provided at 800 µM in DMSO for subsequent serial dilution and use with Trevigen’s HT Universal PARP Assay Kits.

6(5H)-Phenanthridinone Catalog # 4667-50-10 6(5H)-Phenanthridinone strongly inhibits poly (ADP-ribose) polymerase (PARP) and 0023 displays immunosuppressive activity. It is a mixed-type inhibitor that acts on both the enzyme and enzyme-NAD+ complex at site(s) distinct from the NAD+-binding site to attenuate decreases in NAD+ and ATP and, consequently, improve cell survival. It also inhibits concanavalin A-induced lymphocyte proliferation at micromolar concentrations. This inhibitor, which has been analyzed in the range of 0.18-0.39 µM, is provided at 160 µM in DMSO for use with Trevigen’s HT Universal PARP Assay Kits.

Benzamide Catalog # Graphical representation of the chemiluminescent readout of the PARP standard curve and inhibition curve for 3-aminobenzamide using HT 4667-50-11 Universal Chemiluminescent PARP Assay Kit (4676-096-K).Each point Benzamide is the most potent poly (ADP-ribose) polymerase (PARP) inhibitor in the family represents the mean value fro triplicate determinations. of benzamides. It also acts as a neuroprotectant by inhibition of PARP. Benzamide is twice as active as its commonly used counterpart 3-aminobenzamide, in delaying or suppressing PARP activation. It is able to prevent nuclear fragmentation and apoptotic-body formation without affecting DNA fragmentation during apoptosis. This PARP inhibitor, which has been analyzed in the range of 100 to 500 µM, is provided at 8 mM in ethanol for use with Trevigen’s PARP Assay Kits.

DEA ORDERING INFORMATION Catalog # 4680-096-03 4667-50-03 DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) is provided at 100 mM in DMSO 3-Aminobenzamide, 60 µl as a control PARG inhibitor. DEA will inhibit the activity of PARG at a wide range of 4667-50-09 concentrations from 1 µM to 1 mM. This inhibitor is a component of the HT Universal 4-Amino-1,8-naphthalimide, 100 µl PARG Assay Kits. 4667-50-10 6(5H)-Phenanthridinone, 100 µl 4667-50-11 Benzamide, 100 µl 4680-096-03 DEA, 200 µl

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DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® CometAssay® Reagent Kits for Single Cell Gel Electrophoresis Assay A Trevigen’s CometAssay®, or single cell gel electrophoresis assay, provides a simple and effective method for evaluating DNA damage in cells. The principle of the assay is based upon the ability of denatured, cleaved DNA fragments to migrate out of the nucleoid under the influence of an electric field, whereas undamaged DNA migrates slower and remains within the confines of the nucleoid when a current is applied. Evaluation of the DNA “comet” tail shape and migration pattern allows for assessment of DNA damage. The Neutral CometAssay® is typically used to detect double-stranded breaks, whereas the Alkaline CometAssay® is more sensitive, and is used to detect smaller amounts of damage including single and double-stranded breaks.

ASSAY DESIGN FOR ALKALINE COMET: Step 1: Cells mixed with low melting agarose are immobilized on specially treated slides and lysed. • Membranes and histones removed from the DNA of individually isolated cells. Step 2: Samples are treated with alkali to unwind and denature the DNA. B • For detection of single and double-stranded breaks. Step 3: Following alkaline electrophoresis slides are stained and visualized by fluorescent microscopy. • Increase in DNA damage results in smaller fragments migrating faster in an electric field. • Quantitative measurements of DNA damage REAGENT KITS APPLICATIONS: 24 • Applicable for the analysis of either single strand or double strand DNA breaks • Study cellular responses to DNA damage • Screen for inhibitors of DNA repair • Test chemicals for toxicity • Screen for environmental mutagens • Probe for specific types of DNA damage using DNA repair glycosylases Results of a typical CometAssay® experiment in alkali electrophoresis conditions. DNA fragmentation associated with oxidative DNA damage was visualized using the COMPONENTS: CometAssay® kit. (A) Damaged cell showing characteristic Catalog # Description migration of DNA. (B) Untreated cell showing no DNA damage. 4250-050-01 Lysis Solution 4250-050-02 Comet LMAgarose 4250-050-03 CometSlide™ (2 well) 4250-050-04 200 mM EDTA 4250-050-05 SYBR® Green ORDERING INFORMATION RELATED PRODUCTS: 4250-050-01 CometAssay® Electrophoresis System ® CometAssay Lysis Solution, CometAssay® Control Cells 2x500 ml CometAssay® Silver Staining Kit 4250-050-02 CometSlide™ 2, 20 and 96 Well Slides CometAssay® LMAgarose, CometSlide™ Rack System 15 ml FLARE™ Assays and FLARE™ Slide (3 well) 4250-050-05 SYBR® Green, 5 µl STORAGE: Store components at -20˚C, 4˚C, and room temperature. 4250-050-K CometAssay® Kit, 50 Samples (twenty-five 2 well slides) 4252-040-K CometAssay® Kit, 40 Samples (two 20 well slides) 4253-096-K CometAssay® Kit, 96 Samples (one 96 well slide)

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DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® CometAssay® Reagent Kit for Single Cell Gel Electrophoresis Assay

REFERENCES: 1. Lemay, M. and K.A. Wood. 1999. Detection of DNA damage and identification of UV-induced photoproducts using the CometAssay™ kit. BioTechniques 27:846-851. 2. Singh, N.P., R.E. Stephens, and E.L. Schneider. 1994. Modifications of alkaline microgel electrophoresis for sensitive detection of DNA damage. Int J Radiat Biol 66:23-28. 3. Collins, A.R., S.J. Duthie, and V.L. Dobson. 1993. Direct enzymatic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis 14:1733-1735. 4. Collins, A.R. 2004. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol 26:249-261. 5. Olive, P.L. 2002. The comet assay. An overview of techniques. Methods Mol Biol 203:179-194. 6. Horvathova, E., M. Dusinska, S. Shaposhnikov, and A.R. Collins. 2004. DNA damage

and repair measured in different genomic regions using the comet assay with fluorescent OVERVIEW REAGENT KITS in situ hybridization. Mutagenesis 19:269-276. 7. Hartmann, A., M. Schumacher, U. Plappert-Belbig, P. Lowe, W. Suter, and L. Mueller. 2004. Use of the alkaline in vivo comet assay for mechanistic genotoxicity investigations. Mutagenesis 19:51-59.

SYBR® GREEN I NUCLEIC ACID GEL STAIN LICENSING TERMS: This product is sold under license from Molecular Probes, Inc. under US Patents Nos. 5,436,134 and 5,658,751 for use in a comet assay for internal research and development only, where research and development use expressly excludes the use of this product for providing medical, 0025 diagnostic or any other testing analysis or screening services or providing clinical information or clinical analysis, in return for compensation on a per-test basis, and research and development use expressly excludes incorporation of this product into another product for commercialization even if such other product would be commercialized for research and/or development use.

1. Cells mixed with low melting agarose at 37oC (LM Agarose) 2. Immobilize cells on CometSlide™ 3. Treat cells with Lysis Solution (removes membranes and histones from the DNA) 4. Samples treated with alkali (unwinds and denatures DNA) 5. Samples stained with intercalating dye and visualized by epifluorescence microscopy following alkaline electrophoresis, which reveals DNA breaks

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DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® CometAssay® Silver Staining Kit

A Trevigen’s CometAssay® Silver Staining Kit is designed for the convenient silver staining of CometAssay® or single cell gel electrophoresis samples. Recording of data generated by the CometAssay® and staining with fluorescent dyes requires epifluorescence microscopy. Using the Silver Staining Kit, permanent records are created and visualization using standard light microscopy is possible. The Silver Staining Kit was designed specifically for use with the CometSlides™ to minimize unwanted background and the amount of hazardous waste generated by silver nitrate.

FEATURES: • Allows visualization by standard light microscopy • Permanent staining for archiving samples • Optimized for CometSlide™ staining

APPLICATIONS: A CometAssay®

COMETASSAY® SILVER STAINING KIT COMPONENETS Catalog # Component 4254-200-01 20X Staining Reagent #1 4254-200-02 20X Staining Reagent #2

SILVER STAINING KIT STAINING SILVER 4254-200-03 20X Staining Reagent #3 4254-200-04 2X Staining Reagent #4 26 4254-200-05 10X Fixation Additive COMETASSAY® SILVER COMPONENTS Catalog # Component 4250-050-01 Lysis Solution ® Comets visualized using either SYBR Green (A) or 4250-050-02 Comet LMAgarose Trevigen’s CometAssay® Silver Staining Kit (B). Potassium Permanganate treated WEHI 7.1 cells were processed using 4250-050-03 CometSlide™ the CometAssay® Kit. Samples were fixed in cold ethanol 4250-050-04 200 mM EDTA and air dried. Following drying, the slides were stained with 4254-200-K CometAssay® Silver Staining Kit SYBR® Green. Subsequently, the slides were stained with the CometAssay® Silver Staining Kit as recommended in the instructions. STORAGE: • Catalog # 4254-200-K is stored room temperature. Once reconstituted 2X Staining Reagent #4 is stable for 3 months at 4°C.

• Catalog # 4251-050-K has multiple storage temperatures – room, 4°C, -20°C.

REFERENCES: 1. Black, J.A. 1985. A silver stain for isoelectric focusing in agarose gel and its application for analyzing unconcentrated cerebrospinal fluid. Electrophoresis 6:27-29. 2. Delincee, H. 1997. Silver staining of DNA in the “comet assay”. Comet Newsletter No. 6. Kinetic Imaging Inc. Liverpool, UK. ORDERING INFORMATION 3. Gottlieb, M. and M. Chavko. 1987. Silver staining of native and denatured eucaryotic DNA in agarose gels. Anal Biochem 165:33-37. 4250-050-01 CometAssay® Lysis Solution, 2x500 ml 4250-050-02 CometAssay® Silver LMAgarose, 15 ml 4251-050-K CometAssay® Silver (CometAssay® Kit w/Silver Staining Reagents), 50 Samples 4254-200-K CometAssay® Silver Staining Kit, 200 Samples

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DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® CometAssay® Electrophoresis System

Trevigen’s CometAssay® Electrophoresis System enables investigators to consistently optimize alkaline and neutral comet assays for highly reproducible results, and to standardize electrophoresis methods between individual users and laboratories. Comet assay results can be variable depending on temperature, distance between electrodes, and buffer height. Trevigen has solved these problems by developing a specialized electrophoresis unit.

FEATURES: • Maintains constant buffer temperature with cooling water chamber and

tempered glass platform. ELECTROPHORESIS SYSTEM • Maintains optimal buffer level for consistent results with overlay. • Specially designed trays accommodate 2, 20 and 96 well slides and maintain correct position during electrophoresis. • Optimized for use with CometAssay Kits and CometAssay Control Cells. • Available with area specific power supply. OVERVIEW

0027

ORDERING INFORMATION

4250-050-ES CometAssay® ES Unit 4250-050-ESK CometAssay® ES Starter Kit with 2 well slides

4250-050-ESK-PS1 CometAssay® ES Starter Kit with 2 well slides and power supply for North America*

4252-040-ESK CometAssay® ES Starter Kit with 20 well slides 4252-040-ESK-PS1 CometAssay® ES Starter Kit with 20 well slides and power supply for North America* 4253-096-ESK CometAssay® ES Starter Kit with 96 well slides 4253-096-ESK-PS1 CometAssay® ES Starter Kit with 96 well slides and power supply for North America* *International version – alternative power supply for EU, United Kingdom, and Switzerland (see page 189).

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DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® CometAssay® Electrophoresis System

COMETASSAY® ELECTROPHORESIS SYSTEM STARTER KIT Trevigen makes available a complete assay system which includes CometAssay® kits, CometSlides®, Comet Assay® control cells and a specialized electrophoresis unit for alkaline and neutral comet assays.

STARTER KIT (ESK) COMPONENTS

Catalog # Component 4250-05-ES CometAssay® Electrophoresis System 4250-050-K CometAssay® Kit (2 well) 4250-040-K CometAssay® Kit (20 well) 4250-096-K CometAssay® Kit (96 well) 4256-019-CC CometAssay® Control Cells for alkaline comet

Starter Kits available with power supply ELECTROPHORESIS SYSTEM

ALKALINE COMETASSAY® Examples of data collected for each CometAssay® Control Cell population (catalog # 4256-010-CC see page 29).

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DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® CometAssay® Control Cells

Trevigen has developed two sets of suspension cell preparations containing different levels of DNA damage to be used as controls with Trevigen’s CometAssay® Kits when performing alkaline or neutral comet, respectively. These cryopreserved controls are designed to act as controls to standardize and compare comet assay results between individual users and laboratories.

CometAssay® CONTROL CELLS FOR ALKALINE COMET Healthy control cell population (CC0) treated to increase the amount of single-strand damage CometAssay in populations CC1, CC2 and CC3, respectively.

NEUTRAL CometAssay® CONTROL CELLS FOR NEUTRAL COMET Healthy cell population (NC0) treated to increase the amount of double-strand damage in populations NC1, NC2 and NC3, respectively. ® CONTROL CELLS FEATURES: OVERVIEW • Provides a range of DNA damage for comparison with statistically distinct populations. • Allows for electrophoresis method to be standardized between individual users. • Standardized with Trevigen’s CometAssay® Electrophoresis System • Cell populations remain stable in liquid nitrogen for long term storage.

KIT CONTENTS:

Description Size 0029 Healthy Cells 500µl Treated Cells–level 1 500µl Treated Cells–level 2 500µl Treated Cells–level 3 500µl

30 % DNA in Tail

-10 CCO CC1 CC2 CC3 ORDERING INFORMATION Control Cells

4256-010-CC CometAssay® Control Cells (Alkaline), 10 assays Box-Whisker Plot Data collected for each alkaline CometAssay® Control Cell population (catalog # 4256-010-CC) is shown as side-by side vertical box plots for comparison. The diamond shows the mean and confidence interval around the mean. The notched box shows 4257-010-CC the median, lower and upper quartiles, and the 75% confidence interval around the median. Neutral CometAssay® Control Cells, 10 assays

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DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® CometAssay® Slides and Rack System

CometSlide™ 2, 20 and 96 Wells FLARE™ Slide 3 Wells CometSlide™ Rack System

Trevigen's CometSlides™ can be purchased separately and are available in 2, 20, and 96 well format to meet your screening needs. CometSlides™ greatly simplify the comet assay by providing a sample surface specially treated to promote agarose adherence and a hydrophobic barrier to allow treatment with one of Trevigen’s DNA repair enzymes. Trevigen’s 3 well FLARE™Slide can also be interchanged with the CometSlide™. Simply add your cells to low melting point Comet LMAgarose, and pipet onto the slide. The CometSlide™ Rack System was designed to accommodate 2, 3 or 20 well slides.

FEATURES: • Allows the rapid and reliable analysis of large numbers of samples. • CometSlides™ allow direct application of Comet LMAgarose without base

SLIDES layers or pretreatment. ® • Hydrophobic barrier allows easy application of cells directly to each slide, and treatment with any of several Trevigen DNA repair enzymes (FLARE™). • Designed for use with the CometAssay® Electrophoresis System.

APPLICATIONS:

CometAssay CometAssay®

30 RELATED PRODUCTS: ORDERING INFORMATION CometAssay® Electrophoresis System ® 3950-075-02 CometAssay Control Cells ® FLARE™ Slide (3 well, 25 slides) CometAssay Silver Staining Kit FLARE™ Assays 3950-300-02 FLARE™ Slide (3 well, 100 slides) STORAGE: Store slides at room temperature with desiccant. 4250-004-03 CometSlide™ (2 well, 2 slides)

4250-050-03 CometSlide™ (2 well, 25 slides) 4250-200-03 CometSlide™ (2 well, 100 slides) 4252-040-01 CometAssay®HT Slide (20 well, 2 slides) 4252-200-01 CometAssay®HT Slide (20 well, 10 slides) 4252-500-01 CometAssay®HT Slide (20 well, 25 slides) 4252-02K-01 CometAssay®HT Slide (20 well, 100 slides) 4253-096-03 CometSlide™ (96 well, 1 slide) 4252-040-02 CometSlide™ Rack System

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DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® FLARE™ Assay Kits and Slides

Trevigen’s unique FLARE™ (Fragment Length Analysis using Repair Enzymes) Assays provide the ability to characterize DNA damage in single cells using a variety of DNA repair enzymes in conjunction with Trevigen’s CometAssay® Electrophoresis System. To assess the type of DNA damage induced by a putative mutagen, drug, or treatment regimen, cells are harvested after treatment and immobilized in a layer of low melting point agarose on the FLARE™ Slide FLARE™ Slide. Following electrophoresis, an apparent increase in damage as a result of enzyme addition is indicative of unrepaired but recognized molecular lesions within the DNA.

writing surface

FEATURES: FLARE™ ASSAY KITS/SLIDES • FLARE™ Slides allow direct application of low melting point agarose without base layers or pretreatment for three samples. • Hydrophobic barrier allows easy application of cells directly to slide and treatment with enzyme or vehicle control. • Specific DNA damage detection

specially OVERVIEW treated glass • Convenient format surface APPLICATIONS: • CometAssay® • DNA damage detection

hydrophobic barrier STORAGE: Store components at -80˚C, -20˚C, 4˚C, and room temperature.

0031

Catalog # FLARE™ Assay Damage Recognized Page

Universal PARP Assay Kit** 4040-100-FK Fpg 8-oxoguanine, DNA containing formamidopyrimidine moieties 32 4040-100-FM PARP/Apoptosis Kit** 4130-100-FK hOGG1 8-oxoguanine, DNA containing formamidopyrimidine moieties 33 4130-100-FM

4045-01K-FK Endonuclease III Thymine glycol, 5,6-dihydrothymine, urea, 5-hydroxy-6- 34 4045-01K-FM hydrothymine, 5,6-dihydrouracil, alloxan, 5-hydroxy-6-hydrouracil, uracil glycol, 5-hydroxy-5-methylhydantoin, 5-hydroxycytosine, 5-hydroxyuracil, methyltartonylurea, thymine ring saturated or fragmentation product

4055-100-FK T4-PDG Cis-syn isomers of cyclobutane pyrimidine dimers 36 4055-100-FK

4065-100-FK cv-PDG Cis-syn and trans-syn isomers of cyclobutane pyrimidine dimers 37 4065-100-FM

4100-100-FK UVDE Cyclobutane pyrimidine dimmers, (6-4) photoproducts 35 4100-100-FM

REFERENCES: ORDERING INFORMATION 1. Collins, A.R., V.L. Dobson, M. Dusinska, G. Kennedy, and R. Stetina. 1997. 3950-075-SP The comet assay: what can it really tell us? Mutat Res 375:183-193. FLARE™ Sample Prep, 75 Tests 2. Duthie, S.J. and P. McMillan. 1997. Uracil misincorporation in human DNA detected 3950-075-02 using single cell gel electrophoresis. Carcinogenesis 18:1709-1714. FLARE™ Modules and FLARE™ Kit 3. Kruszewski, M., M. Wojewodzka, T. Iwanenko, A.R. Collins, and I. Szumiel. 1998. Slides, 25 each Application of the comet assay for monitoring DNA damage in workers exposed to chronic low-dose irradiation II base damage. Mutat Res 416:37-57. 3950-300-02 FLARE™ Slides, 100 Slides 4. de Boeck, M., D. Lison, and M. Kirsch-Volders. 1998. Evaluation of the in vitro direct and indirect genotoxic effects of cobalt compounds using the alkaline comet assay. Influence of interdonor and interexperimental variability. Carcinogenesis 19:2021-2029. 1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:13 PM Page 36

DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® Fpg FLARE™ Assay Kit and Module

Trevigen’s Fpg FLARE™ (Fragment Length Analysis using Repair Enzymes) Assay provides the ability to probe for oxidative DNA damage in single cells using E. coli Formamidopyrimidine-DNA Glycosylase (Fpg) in conjunction with Trevigen’s CometAssay® single cell gel electrophoresis kit. The Fpg FLARE™ Assay Kit supplies the enzyme, reagents, and FLARE™ Slides to perform the CometAssay®. The FLARE™ Module only provides the enzyme and buffers for use with the CometAssay®.

COMPONENTS: Fpg FLARE™ Assay Kit Catalog # 4040-100-FK

Catalog # Component Size 4040-100-01 E. coli Fpg 500 Units 3950-040-01 25X FLARE™ Buffer 1 40 ml 3950-010-03 REC™ Dilution Buffer 10 ml 3950-100-04 100X BSA Additive 100 µl 3950-075-02 FLARE™ Slides 25 each 4250-050-01 Lysis Solution 2 x 500 ml 4250-050-02 Comet LMAgarose (LMA) 15 ml 4250-050-05 SYBR® Green Concentrate 5 µl

Fpg FLARE™ Module Catalog # 4040-100-FM Fpg FLARE™ Catalog # Component Size 32 4040-100-01 E. coli Fpg 500 Units 3950-040-01 25X FLARE™ Buffer 1 40 ml 3950-010-03 REC™ Dilution Buffer 10 ml 3950-100-04 100X BSA Additive 100 µl

ORDERING INFORMATION

4040-100-FK Fpg FLARE™ Assay Kit, 75 Samples 4040-100-FM Fpg FLARE™ Module, 100 Samples

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DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® hOGGI FLARE™ Assay Kit and Module

Trevigen’s hOGG1 FLARE™ (Fragment Length Analysis using Repair Enzymes) Assay provides the ability to probe for oxidative DNA damage in single cells using hOGG1 in conjunction with Trevigen’s CometAssay® single cell gel electrophoresis kit. The hOGG1 FLARE™ Assay Kit supplies the enzyme, reagents, and FLARE™ Slides to perform the CometAssay®. The FLARE™ Module only provides the enzyme and buffers for use with the CometAssay®.

COMPONENTS: hOGG1 FLARE™ Assay Kit Catalog # 4130-100-FK

Catalog # Component Size 4130-100-01 hOGG1 100 Units 3950-040-01 25X FLARE™ Buffer 1 40 ml 3950-010-03 REC™ Dilution Buffer 10 ml OVERVIEW

3950-100-04 100X BSA Additive 100 µl hOGGI FLARE™ 3950-075-02 FLARE™ Slides 25 each 4250-050-01 Lysis Solution 2 x 500 ml 4250-050-02 Comet LMAgarose (LMA) 15 ml 4250-050-05 SYBR® Green Concentrate 5 µl

hOGG1 FLARE™ Module Catalog # 4130-100-FM

Catalog # Component Size 4130-100-01 hOGG1 100 Units 0033 3950-040-01 25X FLARE™ Buffer 1 40 ml 3950-010-03 REC™ Dilution Buffer 10 ml 3950-100-04 100X BSA Additive 100 µl

ORDERING INFORMATION

4130-100-FK hOGG1 FLARE™ Assay Kit, 75 Samples 4130-100-FM hOGG1 FLARE™ Module, 100 Samples

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DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® Endonuclease III FLARE™ Kit and Module

Trevigen’s Endonuclease III FLARE™ (Fragment Length Analysis using Repair Enzymes) Assay provides the ability to detect DNA damage in single cells using E. coli Endonuclease III (Thymine Glycol-DNA Glycosylase) in conjunction with Trevigen’s CometAssay® single cell gel electrophoresis kit. The Endonuclease III FLARE™ Assay Kit supplies the enzyme, reagents, and FLARE™ Slides to perform the CometAssay®. The FLARE™ Module only provides the enzyme and buffers for use with the CometAssay®.

COMPONENTS: Endonuclease III FLARE™ Assay Kit Catalog # 4045-01K-FK

Catalog # Component Size 4045-01K-01 E. coli Endonuclease III 1000 Units 3950-040-01 25X FLARE™ Buffer 1 40 ml 3950-010-03 REC™ Dilution Buffer 10 ml 3950-100-04 100X BSA Additive 100 µl 3950-075-02 FLARE™ Slides 25 each 4250-050-01 Lysis Solution 2 x 500 ml 4250-050-02 Comet LMAgarose (LMA) 15 ml 4250-050-05 SYBR® Green Concentrate 5 µl

Endonuclease III FLARE™ Module Catalog # 4045-01K-FM ENDONUCLEASE III FLARE™ Catalog # Component Size 34 4045-01K-01 E. coli Endonuclease III 1000 Units 3950-040-01 25X FLARE™ Buffer 1 40 ml 3950-010-03 REC™ Dilution Buffer 10 ml 3950-100-04 100X BSA Additive 100 µl

ORDERING INFORMATION

4045-01K-FK Endonuclease III FLARE™ Assay Kit, 75 Samples 4045-01K-FM Endonuclease III FLARE™ Module, 100 Samples

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DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® UVDE FLARE™ Assay Kit and Module

Trevigen’s UVDE FLARE™ (Fragment Length Analysis using Repair Enzymes) Assay provides the ability to probe for DNA damage in single cells using S. pombe UVDE in conjunction with Trevigen’s CometAssay® single cell gel electrophoresis kit. The UVDE FLARE™ Assay Kit supplies the enzyme, reagents, and FLARE™ slides to perform the CometAssay®. The FLARE™ Module only provides the enzyme and buffers for use with the CometAssay®.

COMPONENTS: UVDE FLARE™ Assay Kit Catalog # 4100-100-FK

Catalog # Component Size 4100-100-01 S. pombe UVDE GST-Δ228 100 µl 3951-040-01 25X FLARE™ Buffer 2 40 ml 3950-010-03 REC™ Dilution Buffer 10 ml 3950-100-04 100X BSA Additive 100 µl OVERVIEW UVDE FLARE™ 3950-100-05 100X Cation Solution 100 µl 3950-075-02 FLARE™ Slides 25 each 4250-050-01 Lysis Solution 2 x 500 ml 4250-050-02 Comet LMAgarose (LMA) 15 ml 4250-050-05 SYBR® Green Concentrate 5 µl

UVDE FLARE™ Module Catalog # 4100-100-FM

Catalog # Component Size 0035 4100-100-01 S. pombe UVDE GST-Δ228 100 µl 3951-040-01 25X FLARE™ Buffer 2 40 ml 3950-010-03 REC™ Dilution Buffer 10 ml 3950-100-04 100X BSA Additive 100 µl 3950-100-05 100X Cation Solution 100 µl

ORDERING INFORMATION

4100-100-FK UVDE FLARE™ Assay Kit, 75 Samples 4100-100-FM UVDE FLARE™ Module, 100 Samples

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DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® T4-PDG FLARE™ Assay Kit and Module

Trevigen’s T4-PDG FLARE™ (Fragment Length Analysis using Repair Enzymes) Assay Kit provides the ability to probe for DNA damage in single cells using T4 phage pyrimidine dimer glycosylase (T4-PDG) in conjunction with Trevigen’s CometAssay® single cell gel electrophoresis kit. The T4-PDG FLARE™ Assay Kit supplies the enzyme, reagents, and FLARE™ Slides to perform the CometAssay®. The FLARE™ Module only provides the enzyme and buffers for use with the CometAssay®.

COMPONENTS: T4-PDG FLARE™ Assay Kit Catalog # 4055-100-FK

Catalog # Component Size 4055-100-01 T4-PDG 100,000 Units 3950-040-01 25X FLARE™ Buffer 1 40 ml 3950-010-03 REC™ Dilution Buffer 10 ml 3950-100-04 100X BSA Additive 100 µl 3950-075-02 FLARE™ Slides 25 each 4250-050-01 Lysis Solution 2 x 500 ml 4250-050-02 Comet LMAgarose (LMA) 15 ml 4250-050-05 SYBR® Green Concentrate 5 µl

T4-PDG FLARE™ Module Catalog # 4055-100-FM T4-PDG FLARE™ Catalog # Component Size 36 4055-100-01 T4-PDG 100,000 Units 3950-040-01 25X FLARE™ Buffer 1 40 ml 3950-010-03 REC™ Dilution Buffer 10 ml 3950-100-04 100X BSA Additive 100 µl

ORDERING INFORMATION

4055-100-FK T4-PDG FLARE™ Assay Kit, 75 Samples 4055-100-FM T4-PDG FLARE™ Module, 100 Samples

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DNA DAMAGE AND GENOMIC INSTABILITY

CometAssay® cv-PDG FLARE™ Assay Kit and Module

Trevigen’s cv-PDG FLARE™ (Fragment Length Analysis using Repair Enzymes) Assay Kit provides the ability to probe for DNA damage in single cells using chorella virus pyrimidine dimer glycosylase (cv-PDG) in conjunction with Trevigen’s CometAssay® single cell gel electrophoresis kit. The cv-PDG FLARE™ Assay Kit provides necessary reagents and FLARE™ Slides to perform the CometAssay® along with enzyme and dilution buffers. The FLARE™ Module provides the enzyme along with the dilution buffer for use with the CometAssay®.

COMPONENTS: cv-PDG FLARE™ Assay Kit Catalog # 4065-100-FK

Catalog # Component Size 4065-100-01 cv-PDG 1000 Units 3950-040-01 25X FLARE™ Buffer 1 40 ml cv-PDG FLARE™ 3950-010-03 REC™ Dilution Buffer 10 ml OVERVIEW 3950-100-04 100X BSA Additive 100 µl 3950-075-02 FLARE™ Slides 25 each 4250-050-01 Lysis Solution 2 x 500 ml 4250-050-02 Comet LMAgarose (LMA) 15 ml 4250-050-05 SYBR® Green Concentrate 5 µl

cv-PDG FLARE™ Module Catalog # 4065-100-FM

Catalog # Component Size 0037 4065-100-01 cv-PDG 1000 Units 3950-040-01 25X FLARE™ Buffer 1 40 ml 3950-010-03 REC™ Dilution Buffer 10 ml 3950-100-04 100X BSA Additive 100 µl

ORDERING INFORMATION

4065-100-FK cv-PDG FLARE™ Assay Kit, 75 Samples 4065-100-FM cv-PDG FLARE™ Module, 100 Samples

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Damage Antibodies Summary of DNA Damage Antibodies Chart DNA DAMAGE ANTIBODIES

1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:13 PM Page 43

DNA DAMAGE AND GENOMIC INSTABILITY

DNA Damage Antibodies Anti-phosphorylated Histone H2AX (γ-H2AX) Polyclonal Antibody

Histone H2AX is a 14 kDa ubiquitous member of the H2A histone family that contains an evolutionarily conserved SQ motif at the C-terminus in eukaryotes. Serine 139 within this motif becomes rapidly phosphorylated to yield a form known as γ -H2AX in response to double-strand DNA damage and apoptosis. Phosphorylation reaches half its maximum between 1-3 minutes after DNA damage occurs, and hundreds to several thousand molecules of γ-H2AX are present per double-strand break. This antibody is unique in only detecting double-strand DNA breaks. -14 kDa PHYSICAL STATE: Rabbit serum containing polyclonal antibody raised against synthetic Phosphorylated peptide. Provided at 8 µg/µl in phosphate buffered saline with 0.01% sodium azide. - + Immunoblot of SDS-extracts from Jurkat cells treated with SPECIFICITY:

and without 120 µM etoposide for 4 hours. Samples were Recognizes mammalian, yeast, D. melanogaster, and X. laevis γ-H2AX. OVERVIEW electrophoresed on an 18% Tris-Glycine gel and transferred ANTI- onto a PVDF membrane. γ-H2AX was detected with STORAGE: anti-phosphorylated histone H2AX antibody followed by anti-rabbit conjugated to horseradish peroxidase Freeze in working aliquots at -20°C to avoid repeated freeze-thawing. γ

and chemiluminescence. -H2AX APPLICATIONS: Suitable for Western Blot Analysis and immunocytochemistry. For Western blotting, a starting dilution of 1:1,000 is recommended, whereas for IC, a starting dilution of 1:100 is recommended. 0039 REFERENCES: 1. Mahadevaiah, S.K., J.M. Turner, F. Baudat, E.P. Rogakou, P. de Boer, J. Blanco-Rodriguez, M. Jasin, S. Keeney, W.M. Bonner, P.S. Burgoyne. 2001. Recombinational DNA double-strand breaks in mice precede synapsis. Nature Gen 27:271-6. 2. Rogakou, E.P., W.Nieves-Neira, C.Boon, Y.Pommier, and W.M.Bonner. 2000. Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. J Biol Chem 275:9390-5. 3. Rogakou, E.P., C. Boon, C. Redon, W.M. Bonner. 1999. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146:905-16. 4. Rogakou, E.P., D.R. Pilch, A.H. Orr, V.S. Ivanova, W.M. Bonner. 1998. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273:5858-68.

ORDERING INFORMATION

4411-PC-020 Anti-Phosphorylated Histone H2AX Human cancer (NCI/ADR) cells were irradiated with 2 Mitotic M. muntjak normal skin fibroblasts were irradiated to (γ-H2AX) Antibody, 20 µl Gy to introduce ds DNA breaks. After fixation introduce ds DNA breaks. After fixation and permeabilization, 4411-PC-100 and permeabilization, cells were labeled with cells were labeled with anti-phosphorylated histone H2AX anti-phosphorylated histone H2AX antibody followed antibody and an anti-rabbit fluorescein conjugate followed by Anti-Phosphorylated Histone H2AX γ by an anti-rabbit fluorescein conjugate. Photo courtesy counterstaining with propidium iodide. Photo courtesy of ( -H2AX) Antibody, 100 µl of Dr. E. Rogakou, National Cancer Institute, Dr. W.M. Bonner, National Cancer Institute, NIH, Bethesda, MD NIH, Bethesda, MD. 1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:13 PM Page 44

DNA DAMAGE AND GENOMIC INSTABILITY

DNA Damage Antibodies Anti-8-oxo-dG Monoclonal Antibody (Clone 2E2)

8-hydroxy-2’-deoxyguanosine (8-oxo-dG) is a modified nucleoside, which is the most ABcommonly studied and detected by-product of DNA damage, caused by oxidative radicals associated with inflammation, carcinogenesis, Parkinson’s and Alzheimer’s diseases, and also aging. 8-oxo-dG can serve as a sensitive indicator of physiological and environmental damage to DNA. This mouse monoclonal antibody is provided to enable the detection of 8-oxo-dG by ELISA, immunohistochemistry, or by immunocytochemistry.

IMMUNOGEN: 8-oxo-dG-conjugated-KLH Figure 1: H2O2 treated (A) and untreated (B) MCF-10A cells stained with 8-oxo-dG antibody (Catalog # 4354-MC-050) according to Trevigen’s pro-vided protocol, which uses an SOURCE: AlexaFluor 488 conjugated anti-mouse antibody. Mouse

SPECIFICITY: This mouse monoclonal antibody specifically binds to 8-hydroxy-2’- deoxyguanosine within DNA

ISOTYPE: 8-oxo-dG Monoclonal Antibody Staining of a Rat Thymus Section IgG2b.

PREPARATION: This antibody is provided as purified immunoglobulin from mouse ascites in 1X PBS containing ANTI-8-OXO-dG 0.01% sodium azide.

40 APPLICATIONS: • ELISA • Immunocytochemistry • Immunohistochemistry

STORAGE: Freeze in working aliquots at -20°C in a manual defrost freezer to avoid repeated freeze-thawings.

Figure 2. Paraffin embedded rat thymus sections were stained with 8-oxo-dG monoclonal antibody (clone 2E2) (Catalog # 4335-MC- REFERENCES: 100) at a 1:250 dilution and detected by Alexa-488, which appears 1. Soultanakis RP, Melamede RJ, Bespalov IA, Wallace SS, Beckman KB, Ames BN, Taatjes DJ, fluorescent green. 8-oxo-dG is present in the cytoplasm and Janssen-Heininger YMW. (2000) Flourescence detection of 8-oxoguanine in nuclear and mitochondrial nucleus of most but not all cells. Sections were counter-stained DNA of cultured cells using a recombinant Fab and confocal scanning laser microscopy. Free Rad with 7-Aminoactinomycin D (7AAD), which labels nuclear DNA. Images were captured at 40X magnification. Biol Med 28:987-998.

ORDERING INFORMATION

4354-MC-050 Anti-8-oxo-dG Monoclonal Antibody (clone 2E2), 50 µl

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Damage Antibodies Anti-FEN-1 Polyclonal Antibody

Rabbit polyclonal antibody to mammalian flap-endonuclease (FEN-1). It is involved in DNA double-strand break repair and base excision repair. This antibody has been shown to inhibit FEN-1 activity.

IMMUNOGEN: Full-length recombinant human FEN-1 protein.

PHYSICAL STATE: This antibody is provided as rabbit anti-sera.

SPECIFICITY: The antibody detects human and mouse FEN-1.

STORAGE: Stable for at least 1 year at -20°C in a manual defrost freezer. Avoid repeated freezing and OVERVIEW thawing by aliquoting into microtubes and storing at -20°C or -80°C. ANTI-FEN-1 APPLICATIONS: For Western Blot Ananlysis, an antibody dilution of 1:1000 is suggested, but empirical testing will be required for optimal results.

NOTICE: The supply of this product by Trevigen conveys to you only a limited, non-transferable Western blot analysis of Wehi and Jurkat cells using right to use this product (or materials made from this product) for purposes other than 0041 Trevigen’s anti-FEN-1 polyclonal antibody. Cells were lysed in Tris-Glycine SDS sample buffer at the concentration therapeutic and prophylactic applications and/or products, including, but not limited to drug 1 x 107 cells/ml and 10 µl of each lysate were loaded per screening or development. Should you desire to use this product (or materials made from well of a 4-20% Tris-Glycine gel. Proteins were transferred this product) for therapeutic or prophylactic applications or products, please contact onto an Immobilon FL membrane and proteins were Athersys, Inc., Cleveland, Ohio, U.S.A. (www.athersys.com). detected with Trevigen’s anti-FEN-1 antibody (catalog # 4410-PC-100) followed by an IR680-conjugated secondary antibody (Licor). Membranes were scanned using an REFERENCES: Odyssey Infrared Imaging System (Licor). 1. Harrington, J.J. and M.R. Lieber. 1994. The characterization of a mammalian DNA structure-specific endonuclease. EMBO 13:1235-1246. 2. Harrington, J.J. and M.R. Lieber. 1994. Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair. Genes and Development 8:1344-1355.

ORDERING INFORMATION

4410-PC-100 Anti-FEN-1 Polyclonal Antibody, 100 µl

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Damage Antibodies Anti-BPDE Antibody (Clone 8E11)

Benzo[a]pyrene diol epoxide

The antibody recognizes both free polycyclic aromatic hydrocarbons (PAHs) and DNA adducts. In an inhibition assay, the binding of the antibody to BPDE adducts was inhibited by BPDE-I-DNA, BPDE-I-dG, BPDE-I tetraol but not by BPDE-II-DNA1.

IMMUNOGEN: The antibody was raised against BPDE-I-G coupled to BSA.

PHYSICAL STATE: This antibody is provided from mouse ascites, provided at 1 mg/ml in PBS, 0.01% sodium azide.

IG CLASS:

IgG1/κ

STORAGE: Stored at 4°C. For long term storage, freeze in working aliquots at -20°C in a manual defrost freezer to avoid repeated freeze-thawings.

APPLICATIONS:

ANTI-BPDE Competitve ELISA and immunoprecipitation. The antibody may be used to investigate exposure to environmental and occupational pollutants. PAHs are released into the 42 environment following incomplete combustion of organic materials. Human exposure to PAHs comes from various occupational, environmental, dietary and medicinal sources. Exposure to this group of compounds is believed to be carcinogenic.

REFERENCES: 1. Santella, R.M., C.D. Lin, W. L. Cleveland, and I.B. Weinstein. 1984. Monoclonal antibodies to DNA modified by a benzo[a]pyrene diol epoxide. Carcinogenesis 5:373-377. 2. Lee, B.M. and R. Santella. 1988. Quantitation of protein adducts as a marker of genotoxic exposure: immunologic detection of benzo[a]pyrene-globin adducts in mice. Carcinogenesis 9:1773-1777. 3. Strickland, P.T., D. Kang, E.D. Bowman, A. Fitzwilliam, T.E. Downing, N. Rothman, J.D. Groopman, and A. Weston. 1994. Identification of 1-hydroxypyrene glucuronide as a major pyrene metabolite in human urine by synchronous fluorescence spectroscopy and gas chromatography-mass spectrometry. Carcinogenesis 15:483-487. 4. Kang, D.H., N. Rothman, M.C. Poirier, A. Greenberg, C.H. Hsu, B.S. Schwartz, M.E. Baser, J.D. Groopman, A. Weston, and P.T. Strickland. 1995. Interindividual differences in the concentration of 1-hydroxypyrene-glucuronide in urine and polycyclic aromatic hydrocarbon- DNA adducts in peripheral white blood cells after charbroiled beef consumption. Carcinogenesis 16:1079-1085. 5. Kang , D.H., N. Rothman, S.-H. Cho, H.S. Lim, H.-J. Kwon, S.-M. Kim, B. Schwartz, and P.T. Strickland. 1995. Association of exposure to polycyclic aromatic hydrocarbons (estimated from job category) with concentration of 1-hydroxypyrene glucuronide in urine from workers at a steel plant. Occupational and Environmental Medicine 52:593-599.

ORDERING INFORMATION

4360-MC-100 Anti-BPDE Monoclonal Antibody (clone 8E11), 100 µg

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Damage Antibodies Anti-UVssDNA Antibody (Clone C3B6)

DNA exposed to UV light accumulates a number of DNA photoproducts, predominantly (6-4) photoproducts and cyclobutane pyrimidine dimers. Isolated DNA may be immobilized in 96 well microplates for analysis by ELISA. Intact cells or tissues may also be investigated using fluorescent immunohistochemical methods. The antibody is ideal for studies involving UV damage to skin or UV damage DNA repair mechanisms.

IMMUNOGEN: The antibody was raised against UV irradiated calf thymus ssDNA conjugated to methylated BSA (mBSA) and UV irradiated poly dT-mBSA complex.

PHYSICAL STATE: The antibody is provided at 1 mg/ml as purified ascites containing 0.01% sodium azide.

IG CLASS: OVERVIEW

IgG1/κ ANTI-UVssDNA DNA isolated from calf thymus was irradiated using a UV light source at either 254 or 312 nm, immobilized onto the bottom SPECIFICITY: of a 96 well microplate, and analyzed by ELISA using the anti- Specificity has been demonstrated for (6-4)-dithymidine, whereas the (6-4)-dicytidines or UVssDNA antibody. cyclo-butadipyrimidines are not recognized. A low level of cross reactivity was observed with repeating (6-4)-thymine/cytidines within an oligonucleotide sequence. The UVssDNA clone C3B6 antibody recognizes (6-4)-dipyrimidines in single stranded DNA at least 4 nucleotides long.

STORAGE: 0043 Store at 4˚C. For long term storage, freeze in working aliquots at -20˚C in a manual defrost freezer to avoid repeated freeze-thawings.

APPLICATIONS: ELISA and indirect in situ immunofluorescence to facilitate the study of the biological significance of DNA lesions. Related products include UVDE (catalog # 4100-100-EB see page 51), DermaTACS™ (catalog # 4829-30-K see page 128), EpiDerm™ Control Slides (catalog # 4800-30-02 see page 128).

REFERENCES: 1. Liang X, Pickering MT, Cho N-H, Chang H, Volkert MR, Kowalik TF, and Jung JU. 2006. Deregulation of DNA Damage Signal Transduction by Herpesvirus Latency-Associated M2. J. Virol. 80:5862-74. 2. Strickland PT, Creasey JS. 1988. Immunoassay of dithymidine cyclobutane dimers in nanogram quantities of DNA. IARC Sci Publ. 89:341-4. 3. Strickland PT, Nikaido O, Matsunaga T, Boyle JM. 1992. Further characterization of monoclonal antibody indicates specificity for (6-4)-dipyrimidine photoproducts. Photochem Photobiol. 55:723-7. 4. Bruze M, Emmett EA, Creasey J, Strickland PT. 1989. Cyclobutadithymidine induction by solar-simulating UV radiation in human skin: II. Individual responses. J Invest Dermatol. 93:341-4. 5. Lesko SA, Li W, Zheng G, Callahan D, Kaplan DS, Midden WR, Strickland PT. 1989. Quantitative immunofluorescence assay for cyclobutyldithymidine dimers in individual mammalian cells. Carcinogenesis 10:641-6.

ORDERING INFORMATION

4350-MC-100 Anti-UVssDNA Monoclonal Antibody (clone C3B6), 100 µg

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes DNA Repair Enzymes Chart

E. coli Endonuclease III 4 AP sites, thymine glycol, 5,6- Radiation damage, 4045-01K-EB 1000 units 49 E. coli nth protein, dihydrothymine, urea, 5-hydroxy-6- oxidation damage, 4045-05K-EB 5000 units Thymine glycol-DNA glycosylase, hydrothymine, 5,6-dihydrouracil, alloxan, UV damage 5-hydroxy-6-hydrouracil, uracil glycol, TG DNA glycosylase 5-hydroxy-5-methylhydantoin, 5-hydroxycytosine, 5-hydroxyuracil, methylartonylurea, thymine ring saturated or fragmentation product

E. coli Endonuclease IV 1 AP sites, urea, phosphoglycolate ends, AP sites, 4050-100-EB 100 units 56 E. coli nfo protein phosphate blocked 3' ends oxidizing agents (bleomycin, tert-butyl 4050-500-EB 500 units hydroperoxide), alkylating agents (MMS, mitomycin) E. coli Fpg Protein 10 AP sites, DNA containing Radiation damage, 4040-100-EB 500 units 50 E. coli FaPy DNA glycosylase formamidopyrimidine moieties, oxidation damage 4040-500-EB 2500 units 8-oxoguanine E. coli Mismatch Uracil DNA 6 3,N4-ethenocytosine, Uracil mismatches Deamination of cytosine 4125-100-EB 100 units 61 Glycosylase E. coli Uracil-N-Glycosylase 6 DNA containing Uracil Deamination of cytosine 4025-100-EB 100 units 58 E. coli ura-DNA glycosylase, E. coli UNGase E. coli MutY DNA Glycosylase 4 A/G, A/8-oxo-dG mismatches A/G and A/8-oxo-dG mismatches, 4000-500-EB 500 units 62 oxidative damage Human AP Endonuclease 7 AP sites, urea, phosphoglycolate ends, AP sites, base excision repair, 4110-01K-Eb 1000 units 45 hAPE/Ref-1 phosphate blocked 3' ends oxidizing agents (bleomycin, tert-butyl 4110-05K-EB 5000 units

DNA REPAIR ENZYMES DNA REPAIR hydroperoxide), alkylating agents (MMS, mitomycin) Human DNA polymerase ß 8 Double stranded DNA with recessed 3' Filling in gaps in DNA, 4020-100-EB 100 units 46 44 OH group base excision repair 4020-500-EB 500 units 4020-01K-EB 1000 units 4020-100-K Kit (100 units) Human FEN-1 12 DNA branch structures Double-strand break repair, 4120-100-EB 100 units 47 base excision repair Human 8-oxo-Guanine DNA 6 AP sites, DNA containing Radiation damage, 4130-100-EB 100 units 48 Glycosylase formamidopyrimidine moieties, oxidation damage 4130-500-EB 500 units hOGG1 8-oxoguanine Human Ku 70/80 Complex N/A Double strand DNA breaks Non-homologous end joining (NHEJ) 4135-250-01 250 units 65 Ku heterodimer Mouse 3-Methyladenine DNA 9 3-methyladenine, 3-methylguanine, DNA methylation, oxidative damage 4090-100-EB 100 units 60 Glycosylase 7-methylguanine, hypoxanthine, 4090-500-EB 500 units 8-oxo-dG M. thermoautotrophicum TDG 4 T/G, G/G mismatches T/G and G/G mismatches, 4070-500-EB 500 units 63 thermostable thymine mismatch DNA deamination of 5-methylcytosine S. pombe Ultraviolet Damage 5 Cyclobutane pyrimidine dimers, (6-4) UV damage 4100-100-EB 100 µl 51 Endonuclease photoproducts S. solfataricus DNA polymerase IV 15 Translesion synthesis across AP sites, Translesion synthesis 4150-010-EB 10 µg 64 thermophilic Dpo4 polymerase cis-syn thymine dimers, 6-4 PCR amplification of damaged DNA 4150-050-EB 50 µg photoproducts, cisplatinum-and acetyl aminofluorene guanine adducts

T4 Endonuclease V 11 Cis-syn isomers of cyclobutane UV damage 4055-100-EB 100,000 52 T4-Pyrimidine Dimer pyrimidine dimers units Glycosylase/T4-PDG

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes hAPE

Human Apurinic/Apyrimidinic Endonuclease

CONTENTS: Catalog # Description Size 4110-01K-01 hAPE 1000 Units 3900-500-07 10X REC™ Buffer 7 1 ml

Human APE (also referred to as Ref-1) is a 37 kDa multifunctional enzyme. It is involved in both DNA repair and in facilitating the redox state for a number of DNA binding proteins.

SOURCE: Purified from E. coli containing a recombinant plasmid harboring the hAPE gene.

UNIT DEFINITION:

One Unit cleaves 1 pmole of a labeled oligonucleotide probe containing an AP site within an OVERVIEW oligonucleotide duplex in one hour at 37°C.

SPECIFICITY: Human APE is responsible for the repair of apurinic/apyrimidinic (AP) sites in the DNA

base excision repair (BER) pathway. It catalyzes the cleavage of the phosphodiester bond hAPE immediately 5′ to an AP site. APE also functions as a redox factor facilitating the DNA-binding capability of JUN, FOS, AP-1, NF-κB, and other transcription factors. The APE-related DNA repair activity has been demonstrated to be inactivated by phosphorylation, suggesting that this might be the mechanism by which the enzyme 0045 switches between the two functions.

ASSAY CONDITIONS: TM 1X REC Buffer 7 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 10 mM MgCl2), 4 pmole of a labeled AP oligonucleotide, annealed to a complement oligonucleotide, and serial dilutions of enzyme in a 20 µl reaction volume are incubated for 1 hour at 37°C. The cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent cleavage quantified.

STORAGE BUFFER: 10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 0.1 mg/ml BSA, 50% (v/v) glycerol.

STORAGE: Store at -20°C in a manual defrost freezer. For long-term storage, freeze at -80°C in working aliquots. Avoid repeated freeze-thawing.

REFERENCES: 1. Hansen, W.K., W.A. Deutsch, A. Yacoub, Y. Xu, D.A. Williams, and M.R. Kelley. 1998. Creation of a fully functional human chimeric DNA repair protein. J Biol Chem 273:756-762. 2. Yacoub, A., M.R. Kelley, and W.A. Deutsch. 1997. The DNA repair activity of human redox/repair protein APE/Ref-1 is inactivated by phosphorylation. Cancer Res 57:5457-5459. 3. Duguid, J.R., J.N. Eble, T.M. Wilson, and M.R. Kelley. 1995. Differential cellular and subcellular expression of the human multifunctional apurinic/apyrimidinic endonuclease (APE/ref-1) DNA repair enzyme. Cancer Res 55:6097-6102. 4. Lieber, M.R. 2008. The Mechanism of Human Nonhomologous DNA End Joining. ORDERING INFORMATION J Biol Chem 283:1 – 5.

4110-01K-EB Human AP Endonuclease, 1000 Units and 10X REC™ Buffer 7, 1 ml 4110-05K-EB Human AP Endonuclease, 5000 Units and 10X REC™ Buffer 7, 1 ml

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes Human DNA Polymerase βKit

CONTENTS: Catalog # Description Qty 4020-100-01 Beta-polymerase 100 µl 4020-050-02 Beta-pol Control DNA 10 µl 3900-200-08 10X RECTM Buffer 8 1 ml 4020-050-04 Aphidicolin 10 µl 4019-1 REC™ water 1 ml 4018-250 5X REC™ Loading buffer 250 µl

β Human DNA Polymerase β is constitutively expressed in cells and functions by filling in gaps in DNA that are formed following base excision repair. The activity of DNA Polymerase β is not affected by aphidicolin, an inhibitor of DNA polymerases α, δ, and ε.

SOURCE: Purified from E. coli containing a recombinant plasmid harboring the human DNA polymerase β gene.

UNIT DEFINITION: One Unit is the amount of enzyme required to catalyze the incorporation of 1 nmole of dNTP into an acid-insoluble form in 1 hour at 37°C.

HUMAN DNA POLYMERASE SPECIFICITY: The enzyme can fill small gaps (up to 6 nucleotides) and nicks in DNA, catalyze DNA 46 synthesis after base excision repair, and release 5'-terminal deoxyribose phosphate residues from incised AP sites.

ASSAY CONDITIONS: TM 1X REC Buffer 8 (50 mM Tris-Cl (pH 8.8), 10 mM MgCl2, 10 mM KCl, 1.0 mM DTT, 1% glycerol) 50 µM dCTP, 50 µM dGTP, 50 µM dATP, 50 µM α-32P-dTTP, and 100 µg/ml of Activated DNA (Catalog # 4667-50-06 see page 128) in a reaction volume of 100 µl are incubated for 5 min at 37°C.

STORAGE BUFFER: 20 mM Tris-Cl (pH 7.8), 1.0 mM DTT, 1 mM EDTA, 100 mM NaCl, and 50% (v/v) glycerol.

STORAGE: Store at -20°C in a manual defrost freezer.

REFERENCES: 1. Matsumoto, Y. and K. Kim. 1995. Excision of deoxyribose phosphate residues by DNA polymerase β during DNA repair. Science 269:699-702. ORDERING INFORMATION 2. Kunkel, T.A. and P.S. Alexander. 1986. The base substitution fidelity of eukaryotic DNA polymerases. J Biol Chem 261:160-166. 4019-1 3. Jenkins, T.M., J.K. Saxena, A. Kumar, S.H. Wilson, and E.J. Ackerman. 1992. REC™ Water, 1 ml DNA polymerase β and DNA synthesis in Xenopus oocytes and in a nuclear extract. 4020-100-EB Science 258:475-478. Human DNA Polymerase ß, 100 Units 4. Vens C, E. Dahmen-Mooren, M. Verwijs-Janssen, W. Blyweert, L. Graversen, H. Bartelink, and 10X REC™ Buffer 8, 1 ml and A.C. Begg. 2002. The role of DNA polymerase beta in determining sensitivity to ionizing 4020-500-EB radiation in human tumor cells. Nucleic Acids Res. 30:2995-3004. Human DNA Polymerase ß, 500 Units 5. Bergoglio V, M.J. Pillaire, M. Lacroix-Triki, B. Raynaud-Messina, Y. Canitrot, A. Bieth, M. and 10X REC™ Buffer 8, 2 ml Gares, M. Wright, G. Delsol, L.A. Loeb, C. Cazaux, and J.S. Hoffmann. 2002. Deregulated 4020-01K-EB DNA polymerase beta induces chromosome instability and tumorigenesis. Human DNA Polymerase ß, 1000 Units Cancer Res. 62:3511-4. and 10X REC™ Buffer 8, 5 ml 6. Kedar P.S., S.J. Kim, A. Robertson, E. Hou, R. Prasad, J.K. Horton, and S.H. Wilson. 2002. 4020-100-K Direct interaction between mammalian DNA polymerase beta and proliferating cell nuclear Human DNA Polymerase ß Kit, 100 Units antigen. J Biol Chem. 277:31115-23.

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes FEN-1 Human Flap Endonuclease CONTENTS: Catalog # Description Size 4120-100-01 Human FEN-1 100 Units 4120-100-02 10X BSA Additive 500 µl 3900-500-12 10X REC™ Reaction Buffer 12 1 ml

FEN-1 is a 50 kDa endonuclease/exonuclease that functions in mammalian DNA replication and repair. The enzyme shows similarity to the yeast Rad2 and Rad13 genes. FEN-1 was identified as a necessary component of Okazaki fragment processing and functions on a number of branched DNA structures. It has been shown that FEN-1 specifically associates with PCNA and this binding stimulates FEN-1 up to 50 fold.

SOURCE: Purified from E. coli containing a recombinant plasmid encoding the human FEN-1 protein. OVERVIEW UNIT DEFINITION: One Unit cleaves 10 pmole of a 32P-labeled oligonucleotide flap complex in one hour at 30°C.

SPECIFICITY: FEN-1 FEN-1 catalyzes the nucleotide excision of DNA branch structures formed during replication of the lagging strand of DNA. The branch structures are formed due to the frequent mismatches that occur at the 5'-end of Okazaki fragments. FEN-1 is also involved in the endonucleolytic cleavage of branch structures that are formed during DNA double strand break end-joining. FEN-1 cleaves DNA flap structures one nucleotide distal to the elbow in 0047 the single-stranded region, or one nucleotide proximal to the elbow in the double-stranded region, creating two possible cleavage products (see figure overleaf). FEN-1 also has a 5'-3' exonuclease activity specific for recessed 5' ends. This exonuclease activity may be involved in the removal of initiator RNA of mammalian Okazaki fragments.

ASSAY CONDITIONS: The flap substrate is prepared by hybridizing 100 pmole Flap Oligonucleotide labeled with 32P, 200 pmole Bridge Oligonucleotide, and 200 pmole Adjacent Oligonucleotide in 20 mM Tris (pH 7.4),15 mM NaCl in a 100 µl volume at 99°C for 10 minutes and then cooling slowly to 4°C. 1X REC™ Reaction Buffer 12, 1X BSA Additive (0.1 mg/ml BSA, 5% glycerol), 8 µl of the above 32P flap substrate, and serial dilutions of enzyme in a reaction volume of 20 µl are incubated for 1 hour at 30°C. For analysis, 5 µl of 5X REC™ Loading Buffer (Cat# 4018-250: 20 mM EDTA, 20% Ficoll, and 0.2% bromophenol blue) are added, and cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis. The bands are cut out and radioactivity counted to quantify the cleavage products.

STORAGE: Store at -20°C in a manual defrost freezer.

STORAGE BUFFER: 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM DTT, 0.1 mg/ml BSA, and 50% (v/v) glycerol.

REFERENCES: 1. Harrington, J.J., and M.R. Lieber. 1994. The characterization of a mammalian DNA structure-specific endonuclease. EMBO Journal 13:1235-1246. 2. Wu, X., J. Li, X. Li, C.-L. Hsieh, P.M.J. Burgers, and M.R. Lieber. 1996. Processing of branched DNA intermediates by a complex of human FEN-1 and PCNA. ORDERING INFORMATION Nucleic Acids Res. 24:2036-2043.

4120-100-EB NOTICE: Recombinant Human FEN-1, 100 Units The supply of this Product by Trevigen conveys to you only a limited, non-transferable right to and 10X REC™ Buffer 12, 1 ml use this Product (or materials made from this Product) for purposes other than therapeutic 10X BSA Additive, 500 µl and prophylactic applications and/or products, including, but not limited to drug screening or development. Should you desire to use this Product (or materials made from this Product) for therapeutic or prophylactic applications or products, please contact Athersys, Inc., 1-800-873-8443 • www.trevigen.com Cleveland, Ohio, U.S.A. (www.athersys.com). INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:14 PM Page 52

DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes hOGG1 Human 8-oxoGuanine DNA Glycosylase

CONTENTS: Catalog # Description Size 4130-100-01 hOGG1 100 Units 3900-500-06 10X RECTM Buffer 6 1 ml

Reactive oxygen species generated from such things as ionizing radiation, cellular metabolism, and chemical genotoxins cause the DNA adducts 7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FaPy). Human 8-oxo-guanine DNA glycosylase (hOGG1) catalyzes the removal of the 8-oxoG and FaPy through cleavage of the DNA phosphodiester bond following Schiff base chemistry. hOGG1 does not recognize the C=O of 8-oxoG as expected, but rather recognizes a proton on N7 of the nucleotide. By mispairing with adenine during replication, 8-oxoG gives rise to G:C to T:A transversions, a frequent somatic mutation in human cancers. In contrast, a FaPy lesion leads to termination of replication and, therefore, is not considered a pre-mutagenic lesion.

SOURCE: Purified from E. coli containing a recombinant plasmid harboring the α-hOGG1 gene (nuclear protein).

UNIT DEFINITION:

hOGG1 One Unit cleaves 1 pmole of a labeled oligonucleotide probe containing 8-oxoG base paired with C within a duplex oligo. 48 SPECIFICITY: The catalytic activity of hOGG1 is dependent upon the base the 8-oxoG is paired with in the order of C>T>G, A. hOGG1 is also catalytically active when FaPy is paired with C. FaPy is only repaired when base paired to cytosine.

ASSAY CONDITIONS AND ANALYSIS: 1X RECTM Buffer 6 (20 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1 mM DTT, 100 µg/ml BSA), 4 pmoles of labeled 8-oxodG oligonucleotide annealed to the compliment oligonucleotide, and serial dilutions of enzyme in a reaction volume of 20 µl are incubated for 1 hour at 37°C. For analysis, 20 µl of 2X Loading Buffer (20 mM EDTA, 95% formamide, and 0.13% bromophenol blue) are added, the samples are heated to 95°C for 10 min then fast cooled to 4°C, and the cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent cleavage quantified.

STORAGE BUFFER: 20 mM Tris-Cl (pH 7.8), 1.0 mM DTT, 1 mM EDTA, 100 mM NaCl, 1 mM DTT and 50% (v/v) glycerol.

STORAGE: Store at -20°C in a manual defrost freezer. For long-term storage, freeze in working aliquots at -80°C to avoid repeated freeze-thawing.

REFERENCES: 1. Bruner, S.D., D.P.G. Norman, and G.L. Verdine. 2000. Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA. Nature 403:859-866. ORDERING INFORMATION 2. Boiteux, S. and J.P. Radicella. 2000. The human OGG1 gene: structure, functions, and its implications in the process of carcinogenesis. Arch Biochem Biophys 377:1-8. 4130-100-EB hOGG1 Protein, 100 Units and 10X REC™ Buffer 6, 1 ml 4130-500-EB hOGG1 Protein, 500 Units and 10X REC™ Buffer 6, 1 ml

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes Endonuclease III E. coli Thymine Glycol-DNA Glycosylase

CONTENTS: Catalog # Description Size 4045-01K-01 Endonuclease III 1000 Units 3900-500-04 10X REC™ Buffer 4 1 ml

Endonuclease III is a DNA glycosylase with an associated AP lyase activity. Endonuclease III releases bases damaged by UV light, ionizing radiation, osmium tetroxide, or acid. It cleaves abasic sites by β-elimination, producing a single nucleotide gap in the DNA, and contains an iron-sulfur group which helps to maintain its three dimensional conformation. The enzyme has a molecular weight of 23.4 kDa and is suitable for use in FLARE™ (see page 31).

SOURCE: ENDONUCLEASE III Purified from E. coli containing a recombinant plasmid harboring the E. coli nth gene. OVERVIEW UNIT DEFINITION: One Unit of enzyme cleaves 1 pmole of an oligonucleotide probe containing an AP site within an oligonucleotide duplex in one hour at 37°C.

SUBSTRATE SPECIFICITY: Endonuclease III catalyzes the excision of the following forms of DNA damage: Cis-trans-thymine glycol, 5,6-dihydrothymine, 5,6-dihydroxydihydrothymine, alloxan, urea, Autoradiograph of E. coli endonuclease III cleavage of a uracil, 5-hydroxy-5-methylhydantoin, methyltartronyl-urea, 6-hydroxy-5,6-dihydro-pyrim- radiolabeled oligonucleotide containing an AP site. E. coli endonuclease III does not cut a THF-containing idines, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxy-6-hydrothymine, 5,6-dihydrouracil, 0049 oligonucleotide duplex (modified AP site). Each lane glycol, 5-hydroxy-6-hydrouracil, and AP sites. contains 0.5 pmol each of the labeled oligonucleotide and its complementary oligonucleotide. Incubation was for ASSAY CONDITIONS & ANALYSIS: 1 hour at 37˚C, and cleavage products were resolved on a 20% polyacrylamide gel containing 8 M urea. Enzyme may be diluted in 10 mM HEPES-KOH (pH 7.4) and 100 mM KCl for immediate use. In 1X RECTM Buffer 4 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 10 mM EDTA), add 4 pmole of a 32P-labeled oligonucleotide containing an AP site, 4 pmole of a complementary oligonucleotide, and serial dilutions of enzyme in a 20 µl reaction volume; incubate for 1 hour at 37°C. The cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis. Bands are cut out and radioactivity counted to quantify the cleavage products.

STORAGE BUFFER: 10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 0.1 mg/ml BSA, and 50% (v/v) glycerol.

STORAGE: Store at –20°C in a manual defrost freezer. For long-term storage, freeze at –80°C in working aliquots. Avoid repeated freeze-thawing.

REFERENCE: 1. Hatahet, Z., Y.W. Kow, A.A. Purmal, R.P. Cunningham, and S.S. Wallace. 1994. New substrates for old enzymes: 5-hydroxycytidine and 5-hydroxy-2’ deoxyuridine are substrates for Escherichia coli endonuclease III and formamido-pyrimidine DNA-N glycosylase while 5-hydroxy-2’-deoxyuridine is a substrate for uracil DNA-glycosylase. J Biol Chem 69:18814-18820.

ORDERING INFORMATION

4045-01K-EB E.coli Endonuclease III, 1000 units and 10X REC™ Buffer 4, 1 ml 4045-05K-EB E.coli Endonuclease III, 5000 units and 10X REC™ Buffer 4, 1 ml

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes Fpg E. coli Formamidopyrimidine-DNA Glycosylase

CONTENTS: Catalog # Description Size 4040-100-01 Fpg 500 Units 3900-500-10 10X REC™ Buffer 10 1 ml

-+ Fpg releases damaged bases preferentially from duplex DNA. It has an associated class I AP lyase activity, leaving both 3' and 5' phosphoryl groups. This results from a β-elimination reaction at the AP sites, producing a single nucleotide gap in the DNA. The enzyme consists of 269 amino acids with a molecular weight of 30.2 kDa.

Intact SOURCE: Purified from E. coli containing a recombinant plasmid harboring the E. coli fpg gene.

UNIT DEFINITION: One Unit cleaves 1 pmole of a labeled oligonucleotide probe containing 8-oxoguanine, within an oligonucleotide duplex in one hour at 37°C.

SPECIFICITY: Fpg catalyzes the excision of the following forms of DNA damage: Cleaved • Open ring forms of 7-methylguanine, including 2,6-diamino-4-hydroxy-5-N-methylfor-

Fpg mamidopyrimidine and 4,6-diamino-5-amidopyrimidine, a lethal lesion. • 8-oxoguanine, a highly mutagenic lesion and probably the most important biological 50 substrate of Fpg. • 5-hydroxycytosine Autoradiograph of E. coli Fpg cleavage of radiolabeled • 5-hydroxyuracil oligonucleotide containing 8-oxo-dG. Each lane contains 0.5 • Aflatoxin-bound imidazole-ring-opened guanine pmol of labeled oligonucleotide and its complementary oligonucleotide. Incubations were for 1 hour at 37˚C with or • Imidazole ring opened N-2-aminofluorene-C8-guanine without 1 unit of Fpg enzyme. Cleavage products were resolved on a 20% polyacrylamide gel containing 8 M urea. ASSAY CONDITIONS & ANALYSIS: 1X REC Buffer 10 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 10 mM EDTA, and 0.1 mg/ml BSA), 4 pmoles of labeled 8-oxo-dG oligonucleotide annealed to the compliment oligonucleotide, and serial dilutions of enzyme in a reaction volume of 20 µl are incubated for 1 hour at 37°C. For analysis, 10 µl of 3X REC Alkali Loading Buffer (Cat# 4017-500: 300 mM NaOH, 97% formamide, and 0.2% bromophenol blue) are added, the samples are heated to 95°C for 10 min then fast cooled to 4°C, and the cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent cleavage quantified.

STORAGE BUFFER: 20 mM Tris-Cl (pH 7.8), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 50% (v/v) glycerol.

STORAGE: Store at -20°C in a manual defrost freezer. For long term storage, freeze in working aliquots at -80°C. Avoid repeated freeze-thawings. May be diluted in 10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 50% glycerol and store at -20°C for up to 1 week. Otherwise, dilute enzyme in 1X REC Buffer 10 and use immediately. It is stable for up to 8 hours at 37°C without any loss in activity.

REFERENCE: ORDERING INFORMATION 1. Tchou, J., V. Bodepudi, S. Shibutani, I. Antoshechkin, J. Miller, A.P. Grollman, and F. Johnson. 1994. Substrate specificity of Fpg protein: recognition and cleavage of 4040-100-EB oxidatively damaged DNA. J Biol Chem 269:15318-15324. E. coli Fpg Protein, 500 Units and 10X REC™ Buffer 10, 1 ml 2. Friedberg, E.C., G.C. Walker, and W. Siede. 1995. DNA Repair and Mutagenesis. American Society of Microbiology, Washington, D.C.: ASM Press. 4040-500-EB 3. Boiteux, S., T.R. O’Connor, and J. Laval. 1987. Formamidopyrimidine-DNA glycosylase of E. coli Fpg Protein, 2500 Units Escherichia coli: cloning and sequencing of the Fpg structural gene and overproduction of and 10X REC™ Buffer 10, 1 ml the protein. EMBO J 6:3177-3183.

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes UVDE S. pombe Ultraviolet Damage Endonuclease

CONTENTS: Catalog # Description Size 4100-100-01 S. pombe UVDE 100 µl 3900-500-05 10X RECTM Buffer 5 500 µl

Ultraviolet Damage Endonuclease (UVDE) is a nuclease involved in the repair of bipyrimidine DNA photo-products and acts as an alternative to base or nucleotide excision repair pathways. Repair of pyrimidine dimers by UVDE-mediated excision occurs by cleavage immediately 5' to the photoproduct site on the DNA strand in an ATP-independent manner, and is more efficient in vitro than nucleotide excision repair.

SOURCE: Trevigen’s UVDE is a fusion protein consisting of glutathione-S-transferase fused to a 123456 truncated form of the UVDE protein from Schizosaccharomyces pombe (GST-Δ228-UVDE). OVERVIEW Unlike full length UVDE, which has proved to be unstable and difficult to solubilize, GST-Δ228-UVDE lacks 228 amino acids from the amino terminus (of UVDE) and is soluble and stable when stored at -80°C. The presence of the N-terminal deletion does not affect the native properties of the protein, and optimal activity occurs at 30°C. N UVDE L SUBSTRATE SPECIFICITY: Enzymatic studies revealed that UVDE from Schizosaccharomyces pombe recognizes S pyrimidine dimers, 6-4 photoproducts, apurinic/apyrimidinic sites, uracil, dihydrouracil, and other non-UV-induced DNA adducts. Biochemical and genetic analysis also suggest that UVDE 0051 may be involved in orchestrating mismatch repair in vivo, and this enzyme is also active on Cutting of UV photoproducts in supercoiled pUC119 insertion-deletion loops. plasmid DNA. Purified supercoiled DNA was exposed to 0 (Lanes 1 and 2), 100 (Lanes 3 and 4), or 1000 J/m2 (Lanes 5 and 6) UV irradiation at 254 nm. The DNA was then ASSAY CONDITIONS & ANALYSIS: incubated in the presence (Lanes 2, 4 and 6) or absence UVDE treatment of supercoiled DNA, containing adducts such as cyclobutane pyrimidine (Lanes 1, 3 and 5) of 250 ng UVDE enzyme for 45 minutes dimers, leads to relaxation of the supercoiling resulting in a relative shift in electrophoretic at 30˚C. DNA was loaded onto a 0.6% TreviGel™ 5000 gel, mobility. This may be visualized in agarose gels by staining with an intercalating dye such as and subjected to electrophoresis. Following electrophoresis, DNA was analyzed by ethidium bromide staining. S indicates ethidium bromide. Qualitative evaluation is performed by comparing the relative amounts of supercoiled, N indicates nicked, and L is linear forms of DNA contained within bands corresponding to supercoiled DNA and to nicked (relaxed) plasmid DNA. forms. The actual concentrations of DNA and enzyme, and incubation times may have to be adjusted for optimal results.

STORAGE BUFFER: 50 mM Tris-Cl (pH 6.0), 10 mM glutathione, and 10% glycerol.

STORAGE: Store at -80°C in working aliquots to avoid repeated freeze-thawing.

REFERENCES: 1. Avery AM, Kaur B, Taylor JS, Mello JA, Essigmann JM, Doetsch PW. 1999. Substrate specificity of ultraviolet DNA endonuclease (UVDE/Uve1p) from Schizosaccharomyces pombe. Nucleic Acids Res 27:2256–64. 2. Kanno S, Iwai S, Takao M, Yasui A (1999) Repair of apurinic/ apyrimidinic sites by UV damage endonuclease; a repair protein for UV and oxidative damage. Nucleic Acids Res 27:3096–103. 3. Kaur B, Fraser JLA. Freyer GA, Davey S, Doetsch PW. 1999. A Uve1p-mediated mismatch repair pathway in Schizosaccharomyces pombe. Mol Cell Biol 19:4703–10. 4. Doetsch, P.W., Beljanski, V., and Song, B. 2006. The ultraviolet damage endonuclease ORDERING INFORMATION (UVDE) protein and alternative excision repair: a highly diverse system for damage recognition and processing. In DNA Damage Recognition, V. Beljanski and B. Song, eds. 4100-100-EB (New York: Taylor & Francis Press), pp. 211–223. UVDE Enzyme, 100 µl and 10X REC™ Buffer 5, 1 ml

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes T4 Endonuclease V T4-Pyrimidine Dimer Glycosylase/T4-PDG

CONTENTS: Catalog # Description Size 4055-100-01 T4 Endonuclease V 100,000 Units 3900-500-11 10X REC™ Buffer 11 1 ml

T4 Endonuclease V is a DNA glycosylase of the MW 16 kDa protein with an associated AP lyase activity that is specific for the cis-syn isomer of cyclobutane pyrimidine dimers induced by UV irradiation. The enzyme cleaves the glycosyl bond, 5’ to the pyrimidine dimer, via nucleophilic attack of the alpha amino group of Thr-2, forming a series of protein-DNA imino intermediates. The associated AP lyase activity results in cleavage of the phosphodiester bond 3’ to the AP site, thus generating a 3’ unsaturated aldehyde and a 5’ phosphate. This site is then incised on the 5’ side by an AP endonuclease, generating an appropriate substrate for resynthesis and DNA repair.

SOURCE: Purified from E. coli containing a recombinant plasmid harboring the T4 phage denV gene.

UNIT DEFINITION: One Unit is the amount of enzyme required to completely relax 250 ng of a UV-irradiated supercoiled plasmid in 30 minutes at 37°C. T4 ENDONUCLEASE V SUBSTRATE SPECIFICITY: 52 T4-PDG recognizes cis-syn cyclobutane pyrimidine dimers and AP sites. The enzyme recognizes trans-syn dimers at less than 1% the efficiency of the cis-syn isomer. T4-PDG also cleaves at 2,6-diamino-4-hydroxy-5-N-methylformamidoadenine (FAPYA) residues but at only 1-3% that of cis-syn cyclobutane pyrimidine dimers.

ASSAY CONDITIONS & ANALYSIS: 1X REC™ Buffer 11 (25 mM sodium phosphate pH 6.8, 1 mM EDTA, 100 mM NaCl, 1 mM DTT, 0.01% Triton X-100); 0.1 mg/ml BSA, supercoiled plasmid (250 ng) irradiated with 100 J/m2 UV light, and serial dilutions of enzyme in a 20 µl reaction volume are incubated for 30 minutes at 37°C. For analysis, 5 µl of 5X REC Loading buffer (20 mM EDTA. 25% Ficoll, and 0.1% bromophenol blue) are added, and the supercoiled, linear, and open circle forms of the plasmid are resolved by 1% agarose gel electrophoresis. Bands are visualized by ethidium bromide staining.

ORDERING INFORMATION

4055-100-EB T4-Pyrimidine Dimer Glycosylase/ T4-PDG (T4 Endonuclease V), 100,000 Units and 10X REC™ Buffer 11, 1 ml

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes T4 Endonuclease V T4-Pyrimidine Dimer Glycosylase/T4-PDG

STORAGE BUFFER: 20 mM HEPES-NaOH pH 7.0, 0.5 M NaCl, 0.1 mM EDTA.

STORAGE: Store at 4°C. Do not freeze enzyme. May be diluted in 1X RECTM Buffer 11 and used immediately. To preclude loss of activity due to adsorption to plastic or glass surfaces, include BSA at 100 µg/ml in all buffers and assays.

REFERENCES: 1. Vassylyev DG, Kashiwagi T, Mikami Y, Ariyoshi M, Iwai S, Ohtsuka E, Morikawa K (1995). Atomic model of a pyrimidine dimer excision repair enzyme complexed with a DNA T4 ENDONUCLEASE V substrate: structural basis for damaged DNA recognition. Cell 83:773-82. 2. Golan G, Zharkow DO, Grollman AP, Dodson ML, McCullough AK, Lloyd RS, Shoham G (2006) Structure of T4 pyrimidine dimer glycosylase in a reduced imine covalent complex OVERVIEW with abasic site-containing DNA. J Mol Biol 362:241-58.

0053

Glycosylase AP Lyase Glycosylase AP Lyase

T4-PDG catalyzes the fisrt step of DNA excision repair by employing two distinct activities, a pyrimidine dimer (PD)-specific glycosylase, which cleaves the glycosyl bond 5’ of the PD, and an apyrimidinic (AP) lyase, which cleaves the phosphodiester bond 3’ of the abasic site through β elimination to produce an α,β -unsaturated aldehyde and a 5’ terminal phosphate.

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes cv-PDG Chlorella Virus Pyrimidine Dimer Glycosylase

CONTENTS: Catalog # Description Size 4065-100-01 cv-PDG 1000 Units 3900-500-11 10X REC™ Buffer 11 1 ml

cv-PDG is a DNA glycosylase of MW 16 kDa with an associated AP lyase activity that is equally specific for the cis-syn and trans-syn isomers of cyclobutane pyrimidine dimers induced by UV irradiation. The enzyme acts through formation of a protein-DNA imino intermediate. The associated AP lyase activity occurs by a β-elimination reaction, resulting in cleavage of the phosphodiester bond 3’ to the AP site and formation of a 3’ unsaturated aldehyde and 5’ phosphate. This site is then incised on the 5’ side by an AP endonuclease, generating an appropriate substrate for resynthesis and DNA repair.

SOURCE: Purified from E. coli containing a recombinant plasmid harboring the Paramecium bursaria Chlorella virus PDG gene.

UNIT DEFINITION: One Unit is the amount of enzyme required to completely relax 250 ng of a UV-irradiated supercoiled plasmid in 30 minutes at 37°C. cv-PDG SUBSTRATE SPECIFICITY: 54 cv-PDG recognizes cis-syn and trans-syn cyclobutane pyrimidine dimers and AP sites with equal efficiency. The enzyme cleaves AP sites on both double and single stranded DNA.

ASSAY CONDITIONS & ANALYSIS: 1X REC™ Buffer 11 (25 mM sodium phosphate (pH 6.8), 1 mM EDTA, 100 mM NaCl, 1 mM DTT, 0.1 mg/ml BSA), supercoiled plasmid (250 ng) irradiated with 100 J/m2 UV light, and serial dilutions of enzyme in a 20 µl reaction volume are incubated for 30 minutes at 37°C. For analysis, 5 µl of 5X REC™ Loading buffer (cat# 4018-250: 20 mM EDTA, 20% Ficoll, and 0.2% bromophenol blue) are added and the supercoiled, linear, and open circle forms of the plasmid are resolved by 1% agarose gel electrophoresis. Bands are visualized by ethidium bromide staining.

ORDERING INFORMATION

4065-100-EB Chlorella Virus Pyrimidine Dimer (cv-PDG), 1000 Units and 10X REC™ Buffer 11, 1 ml

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes cv-PDG Chlorella Virus Pyrimidine Dimer Glycosylase

STORAGE BUFFER: 25 mM sodium phosphate (pH 6.8), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA.

STORAGE: Store at 4°C. Do not freeze enzyme. May be diluted in 1X REC™ Buffer 11 and used immediately. To preclude loss of activity due to adsorption to plastic or glass surfaces, include BSA at 100 µg/ml in all buffers and assays.

REFERENCES: 1. Garvish, J.F. and R.S. Lloyd. The catalytic mechanism of a pyrimidine dimer-specific glycosylase (pdg)/abasic lyase, chlorella virus-pdg. 1999. J Biol Chem 274:9786-9794. 2. R.S. Lloyd. 1999. The initiation of NA base excision repair of dipyrimidine photoproducts. Progress in Nuc Acid Res and Mol Biol 62:155-175. 3. Garvish, J.F. and R.S. Lloyd. Active-site determination of a pyrimidine dimer glycosylase. OVERVIEW 1999. J Mol Biol 295:479-488. cv-PDG

0055

cv-PDG reaction scheme

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes Endonuclease IV E. coli Endonuclease IV Nfo protein

CONTENTS: Catalog # Description Size 4050-100-01 Endonuclease IV 100 Units 3900-500-01 10X RECTM Buffer 1 1 ml

Endonuclease IV is a class II AP endonuclease with no associated N-glycosylase.

SOURCE: Purified from E. coli containing a recombinant plasmid harboring the E. coli nfo gene.

UNIT DEFINITION: One Unit cleaves 1 pmole of a labeled oliognucleotide probe containing an THF within an oligonucleotide duplex in one hour at 37°C.

SPECIFICITY: Endonuclease IV cleaves AP-sites and removes phosphoglycoaldehyde, deoxyribose-5- phosphate, and 4-hydroxy-2-pentanal. It is not stimulated by cofactors such as Mg2+ or Ca2+, but is inhibited by EDTA (suggesting a metal ion cofactor). Endonuclease IV exhibits exonuclease activity at high protein concentrations. The working concentration may need to be optimized by serially diluting the enzyme (a 1:10 dilution is recommended as a good ENDONUCLEASE IV starting point).

56 ASSAY CONDITIONS: 1X RECTM Buffer 1 (10 mM HEPES-KOH, pH 7.4, 100 mM KCl), 0.1 mg/ml BSA, 4 pmole(s) of a labeled THF oligonucleotide, annealed to complement oligonucleotide, and serial dilutions of enzyme in a 20 µl reaction volume are incubated for 1 hour at 37°C. The cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent cleavage quantified.

ORDERING INFORMATION

4050-100-EB E. coli Endonuclease IV, 100 Units and 10X REC™ Buffer 1, 1 ml 4050-500-EB E. coli Endonuclease IV, 500 Units and 10X REC™ Buffer 1, 1 ml

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes Endonuclease IV E. coli Endonuclease IV Nfo protein

STORAGE BUFFER: 50 mM MOPS-NaOH, pH 8.0, 50 mM NaCl, and 50% (v/v) glycerol.

STORAGE: Store at -20°C in a manual defrost freezer. For long term storage, freeze in working aliquots at -80°C. Avoid repeated freeze-thaw cycling.

REFERENCES: 1. Doetsch, P.W. and R.P. Cunningham. 1990. The enzymology of apurinic/ apyrimidinic endonucleases. Mutat Res 236:173-201. 2. Cunningham, R.P., S.M. Saporito, S.G. Spitzer, and B. Weiss. 1986. Endonuclease IV (nfo)

mutant of Escherichia coli. J Bacteriol. 168:1120-27. ENDONUCLEASE IV 3. Friedberg, E.C., G.C. Walker, and W. Siede. 1995 in DNA Repair and Mutagenesis, American Society of Microbiology. Washington, D.C.: ASM Press. OVERVIEW 4. Levin, J.D., R. Shapiro, and B. Demple. 1991. Metalloenzymes in DNA repair: Escherichia coli endonuclease IV and Saccharomyces cerevisiae. J Biol Chem 266:22893-898.

0057

Endo IV reaction scheme

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes UNGase

E. coli Uracil-N-Glycosylase

CONTENTS: Catalog # Description Size 4025-100-01 Uracil-N-Glycosylase 100 Units 3900-500-06 10X REC™ Buffer 6 1 ml

Uracil bases in DNA form by deamination of cytosine, giving rise to C:G to T:A transitions. A known mechanism to correct this DNA base mutation in E. coli utilizes Uracil-N-Glycosylase, a DNA glycosylase that removes uracil to generate an AP site.

SOURCE: Purified from E. coli containing a recombinant plasmid harboring the E. coli ung gene.

UNIT DEFINITION: One Unit cleaves 1 pmole of a labeled oligonucleotide probe containing uracil, within an oligonucleotide duplex in one hour at 37°C.

SPECIFICITY: Uracil-N-Glycosylase hydrolyzes uracil from single-stranded or double-stranded DNA, but not from oligonucleotides with 6 or fewer bases. It also recognizes 5-fluorouracil, 5-hydroxy- UNGase uracil and isodialuric acid.

58 ASSAY CONDITIONS: 1X RECTM Buffer 6 (20 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1 mM DTT, 0.1mg/ml BSA), 4 pmole of labeled uracil oligonucleotide annealed to the compliment oligonucleotide, and serial dilutions of enzyme in a reaction volume of 20 µl are incubated for 1 hour at 37°C. To complete cleavage of abasic site, fresh 1N NaOH is added to final concentration of 166 mM then heated for 15 minutes at 95°C. For analysis, 24 µl of 2X Loading Buffer (20 mM EDTA, 95% formamide, 0.13% bromophenol blue) are added, the samples are heated to 95°C for 10 min then fast cooled to 4 °C, and the cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent cleavage quantified.

ORDERING INFORMATION

4025-100-EB E. coli Uracil-N-Glycosylase (UNGase), 100 Units and 10X REC™ Buffer 6, 1 ml

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes UNGase

E. coli Uracil-N-Glycosylase

STORAGE BUFFER: 20 mM Tris-HCl (pH 8.0), 50% (v/v) glycerol, 50 mM NaCl, 1 mM EDTA, 1mM DTT, and 0.1 mg/ml BSA.

STORAGE: Store at -20°C in a manual defrost freezer. For long term storage, freeze in working aliquots at – 80°C. Avoid repeated freeze-thawings.

REFERENCES: 1. Duncan, B.K., 1981. DNA glycosylases in The Enzymes (Boyer, P.D., ed), pp. 565-586. New York: Academic Press. 2. Friedberg, E.C., G.C. Walker, and W. Siede. 1995. DNA Repair and Mutagenesis. American Society of Microbiology, Washington D.C:ASM Press. OVERVIEW 3. Verri, A., P. Mazzarello, S. Spadari, and F. Focher. 1992. Uracil-DNA glycosylases preferentially excise mispaired uracil. Biochemistry Journal 287:1007-1010. 4. Takeuchi, R., S. Kimura, A Saotome, and K. Sakaguchi. 2007. Biochemical properties of UNGase a plastidial DNA polymerase of rice. Plant Mol Biol 64:601-611. 5. Parlanti, E., G. Locatelli, G. Maga, and E. Dogliotti. 2007. Human base excision repair complex is physically associated to DNA replication and cell cycle regulatory proteins. Nuc Acids Res 35:1569-1577. 0059

UNGase reaction scheme

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Damage Antibodies Aag

Mouse 3-Methyladenine DNA Glycosylase Type II

CONTENTS: Catalog # Description Size 4090-100-01 Aag protein 100 Units 3900-500-09 10X REC™ Buffer 9 1 ml

Mouse Aag is a 36 kDa constitutively expressed (1,000-2,000 copies/cell) protein. It acts on 3-methyladenine, 3-methylguanine, 7-methylguanine, hypoxanthine, and a number of other substrates.

SOURCE: Purified from E. coli containing a recombinant plasmid encoding the mouse Aag protein.

UNIT DEFINITION: One Unit cleaves 1 pmole of a labeled oligonucleotide probe containing hypoxanthine within an oligonucleotide duplex in one hour at 37°C.

SPECIFICITY: Mouse Aag catalyzes the excision of the following forms of DNA damage: 3-methyladenine,

3-methylguanine, 7-methylguanine, hypoxanthine, and 1,N6-ethenoadenine. It may also

Aag function on the following forms of DNA damage: 7- and 3-ethylpurines,1-carboxyethylade-

nine, 7-carboxyethylguanine, O2-methylpyrimidines, 7(2-ethoxyethyl)-guanine, 60 7(2-hydroxyethyl)guanine, 7(2-chloroethyl)guanine, 1,2-bis(7-guanyl)ethane, 3-ethylthioethylpurines, N2,3-ethenoguanine, N2,3-ethanoguanine, 5-hydroxymethyluracil,

5-formyluracil, 3,N4-ethenocytosine, 1,N2-ethenoguanine, 3,N2-etheno-guanine, and chloroac- etaldehyde cyclic adducts.

ASSAY CONDITIONS: 1X RECTM Buffer 9 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 1 mM EDTA, 1 mM EGTA, and 0.1 mM DTT), 4 pmole of labeled hypoxanthine oligonucleotide annealed to the compliment oligonucleotide, and serial dilutions of enzyme in a reaction volume of 20 µl are incubated for 1 hour at 37°C. To complete cleavage of an abasic site, fresh 1N NaOH is added to final concentration of 166 mM then heated for 15 minutes at 95°C. For analysis, 24 µl of 2X Loading Buffer (20 mM EDTA, 95% formamide, and 0.13% bromophenol blue) are added, the samples are heated to 95°C for 10 minutes then fast cooled to 4°C, and the cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent cleavage quantified.

STORAGE BUFFER: 10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA, and 50% (v/v) glycerol.

STORAGE: Store at -20°C in a manual defrost freezer.

REFERENCES: ORDERING INFORMATION 1. Mattes, W.B., C.-S. Lee, J. Laval, and T.R. O’Conner. 1996. Excision of DNA adducts of nitrogen mustards by bacterial and mammalian 3-methyladenine-DNA glycosylases. 4090-100-EB Carcinogenesis 17:643-648. Mouse 3-Methyladenine DNA 2. Samson, L., B. Derfler, M. Boosalis, and K. Call. 1991. Cloning and characterization of Glycosylase (Aag protein), 100 Units a 3-methyladenine DNA glycosylase cDNA from human cells whose gene maps to and 10X REC™ Buffer 9, 1 ml chromosome 16. Proc. Natl. Acad. Sci. USA 88:9127-9131. 4090-500-EB Mouse 3-Methyladenine DNA Glycosylase (Aag protein), 500 Units and 10X REC™ Buffer 9, 1 ml

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Damage Antibodies Mug

E. coli Mismatch Uracil DNA Glycosylase

CONTENTS: Catalog # Description Size 4125-100-01 Mug protein 100 Units 3900-500-06 10X REC™ Buffer 6 1 ml

E. coli Mug is an 18 kD constitutively expressed protein. The Mug protein can remove Intact a uracil base from within a U:G mismatch as well as act on 3, N4-ethenocytosine-G mismatches (eC:G).

SOURCE: Purified from E. coli containing a recombinant plasmid encoding the E. coli Mug protein. Cleaved

UNIT DEFINITION: OVERVIEW One Unit cleaves 1 pmole of a labeled oligonucleotide probe containing 3,N4-ethenocytosine within an oligonucleotide duplex in one hour at 37°C.

SPECIFICITY:

E. coli Mug catalyzes the excision of the following forms of DNA damage: 3,N4 -ethenocyto- Mug In vitro assay for Mug activity: 2 pmole of 3,N4-ethenocytosine in double or single stranded oligonucleotides. It also excises Uracil in Uracil-Guanine 32 sine oligonucleotide was end-labeled with P and annealed mismatches only in double stranded oligonucleotides. with 2 pmoles of complement to generate an eC:G mismatch. The DNA duplex was incubated with 1 unit of Mug (Catalog # 4125-100-01) for 1 hour at 37˚ C. ASSAY CONDITIONS & ANALYSIS: 0061 The reaction was stopped by the addition of Alkaline 1X RECTM Buffer 6 (20 mM Tris-Cl (pH 8.0), 0.1 mg/ml BSA, 1 mM EDTA, and 1 mM DTT), Loading buffer. The products were resolved on a 20% 4 pmole of labeled 3,N4-ethenocytosine oligonucleotide annealed to the complement denaturing polyacrylamide gel and visualized by oligonucleotide, and serial dilutions of enzyme in a reaction volume of 20 µl are incubated autoradiography. The upper bands represent the full length 3,N4-ethenocytosine oligonucleotide and the lower bands for 1 hour at 37°C. To complete cleavage of an abasic site, fresh 1N NaOH is added to indicate cleavage by Mug. final concentration of 166 mM then heated for 15 minutes at 95°C. For analysis, 24 µl of 2X Loading Buffer (20 mM EDTA, 95% formamide, and 0.13% bromophenol blue) are added and the samples heated to 95°C for 10 min then fast cooled to 4°C. The cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent cleavage quantified.

STORAGE BUFFER: 20 mM Tris-Cl (pH 8.0), 2 mM EDTA, 2.5 mM 2-mercaptoethanol, 1 mM PMSF and 50% (v/v) glycerol.

STORAGE: Store at -20°C in a manual defrost freezer.

REFERENCES: 1. Lutsenko, E., and A. S. Bhagwat. 1999. The role of the Escherichia coli mug protein in the removal of uracil and 3,N4-ethenocytosine from DNA. J. Biol. Chem. 274:31034-31038. 2. Saparbaev, M. and J. Laval. 1998. 3,N4-ethenocytosine, a highly mutagenic adduct, is a primary substrate for Escherichia coli double-stranded uracil-DNA glycosylase and human mismatch-specific thymine-DNA glycosylase. Proc. Natl. Acad. Sci. USA 95:8505-8513.

ORDERING INFORMATION

4125-100-EB E. coli Mismatch Uracil DNA Glycosylase (Mug protein), 100 Units and 10X REC™ Buffer 6, 1 ml

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes MutY

E. coli MutY DNA Glycosylase

CONTENTS: Catalog # Description Size 4000-500-01 E. coli MutY DNA Glycosylase 5000 Units 3900-500-04 10X REC™ Buffer 4 1 ml

E. coli MutY acts together with Fpg to prevent the potentially mutagenic consequences of 8-oxo-dG lesions. 8-oxo-dG lesions escaping repair by Fpg frequently pair with A during DNA replication, producing an 8-oxo-dG:A mispair. MutY removes the A from this base pair to initiate base excision repair. In the absence of MutY, DNA replication after an 8-oxo-dG:A mismatch results in thymine incorporation opposite the adenine in one of the daughter strands, creating a fixed mutation. MutY has an associated AP lyase activity.

SOURCE: Purified from E. coli containing a recombinant plasmid harboring the E. coli MutY gene.

UNIT DEFINITION: One unit of enzyme cleaves 1 pmole of an oligonucleotide duplex containing an A/G mismatch in 1 hour at 37 oC. Only the strand with the A is cleaved.

ASSAY CONDITIONS & ANALYSIS: MutY 4 pmoles of A/G mismatch oligonucleotide set with the A oligo end-labeled, 1X REC™ Buffer 62 4 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 10 mM EDTA), and serial dilutions of enzyme in a 20 µl reaction volume are incubated for 1 hour at 37°C. To complete cleavage of an abasic site, fresh NaOH is added to a final concentration of 166 mM then heated for 15 minutes at 95°C. For analysis, 24 µl of 2X Loading Buffer (20 mM EDTA, 97% formamide, and 0.2% bromophenol blue) is added, the samples are heated at 95°C for 5 min then fast cooled to 4°C, and the cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis. The bands are analyzed to quantify the cleavage products.

STORAGE BUFFER: 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and 50% (v/v) glycerol.

STORAGE: Store at -20°C in a manual defrost freezer. For long term storage, freeze in working aliquots at -80°C. Avoid repeated freeze-thawings. Enzyme may be diluted in storage buffer containing 0.1 mg/ml BSA and stored at -20°C for 2 weeks of experimental use.

REFERENCES: 1. Lu, A. and I. Hsu. 1992. Detection of single DNA base mutations with mismatch repair enzymes. Genomics 14:249-255. 2. Friedberg, E.C., G.C. Walker, and W. Siede. 1995. DNA Repair and Mutagenesis. American Society of Microbiology. Washington, D.C: ASM Press. 3. Hsu, I., W.E. Highsmith, J. Xu, and D. Kong. 1998. Mismatch cleavage detects base deletion in cystic fibrosis gene. Biotechniques 25:692-696.

ORDERING INFORMATION

4000-500-EB E. coli Mut Y Enzyme, 500 Units and 10X REC™ Buffer 4, 1 ml

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes Thermostable TDG

Thymine DNA Glycosylase

CONTENTS: Catalog # Description Size 4070-500-01 Thermostable TDG Protein 500 Units 3900-500-04 10X REC™ Buffer 4 1 ml

TDG is a thermostable thymine DNA glycosylase from Methanobacterium thermoautotrophicum. The optimal temperature for the enzyme is 65°C. The enzyme lacks significant AP lyase or endonuclease activity. TDG works effectively in heteroduplex analysis to detect C to T transitions.

SOURCE: Thermostable TDG is purified from E. coli containing a recombinant plasmid harboring the

Methanobacterium thermoautotrophicum TDG gene. OVERVIEW

UNIT DEFINITION: One Unit is the amount of enzyme required to cleave 1 pmole of an oligonucleotide duplex containing a T/G mismatch in 1 hour at 65°C. Only the strand containing the T is cleaved. TDG SUBSTRATE SPECIFICITY: TDG enzyme recognizes T/G mismatches in duplex DNA and cleaves the strand with the T. The opposite strand is not cleaved. The enzyme also recognizes G/G mismatches if at least one nearest neighbor is an A or T and nicks one strand or the other. The enzyme exhibits 0063 poor AP lyase activity.

ASSAY CONDITIONS & ANALYSIS: 4 pmoles of T/G mismatch oligonucleotide set with the T oligo end-labeled, 1X REC™ Buffer 4 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, and 10 mM EDTA), and serial dilutions of enzyme in a 20 µl reaction volume are incubated for 1 hour at 65°C. To complete cleavage of an abasic site, fresh 1N NaOH is added to a final concentration of 166 mM then heated for 15 minutes at 95°C. For analysis, 24 µl of 2X Loading Buffer (20 mM EDTA, 95% formamide, and 0.13% bromophenol blue) are added, and the samples heated at 95°C for 10 min then fast cooled to 4°C. The cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent cleavage quantified.

STORAGE BUFFER: 25 mM Hepes (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 50% (v/v) glycerol.

STORAGE: Store at -20°C in a manual defrost freezer. For long term storage, freeze in working aliquots at -80°C. Avoid repeated freeze-thawing. Enzyme may be diluted in 1X RECTM Buffer 4 for immediate use. TDG protein in storage buffer can survive for up to 24 hours at 37°C with less than 10% loss in activity.

REFERENCES: 1. Horst, J.P. and H.J. Fritz. 1996. Counteracting the mutagenic effect of hydrolytic deamination of DNA 5-methylcytosine residues at high temperature: DNA mismatch N-glycosylase Mig. Myth of thenthermophilic archaeon Methanobacterium thermoautotrophicum. EMBO J 15:5459-5469.

2. Begley, T.J., and R.P.C. Cunningham.1999. Methanobacterium thermoformicicum thymine ORDERING INFORMATION DNAmismatch glycosylase; conversion of an N-glycosylase to an AP lyase. Protein Engineering 12:333-340. 4070-500-EB Thermostable TDG Protein, 500 Units and 10X REC™ Buffer 4, 1 ml

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes Dpo4

Sulfolobus solfataricus DNA polymerase IV

CONTENTS: Catalog # Description Size 4150-050-01 Dpo4 50 µg 3900-500-15 10X RECTM Buffer 15 1 ml 4150-010-02 100 mM MgCl2 1 ml 4150-010-03 50 mM MnCl2 1 ml

Dpo4 is a thermophilic Y-family DNA polymerase capable of performing translesion synthesis and allows for coupling to PCR for amplification of damaged DNA. Dpo4 has a molecular weight of 42 kDa and is 95% pure by SDS PAGE. Translesion synthesis is enhanced by the presence of Mn2+ in the reaction.

SOURCE: Purified from E. coli containing a recombinant plasmid harboring the S. solfataricus Dpo4 gene.

UNIT DEFINITION:

One unit of enzyme incorporates 1 nmole of dTMP onto the 3’ end of d(T)24 using poly(dA)300 as the template in 30 minutes at 60°C. Dpo4 READ-THROUGH SPECIFICITY: 64 Dpo4 catalyzes translesion synthesis across the following forms of DNA damage: AP sites, cis-syn thymine-thymine dimer, 6-4 photoproducts, cisplatinum- and acetyl-aminofluorene guanine adducts.

ASSAY CONDITIONS & ANALYSIS: 1X RECTM Buffer 15 [10 mM HEPES-NaOH (pH 7.4), 1 mM DTT, 50 mM NaCl, 100 µg/ml BSA, 32 0.1% Triton X-100], 10 mM MgCl2, 1 pmole of P-labeled d(T)24, 2 pmole of poly(dA)300, and serial dilutions of enzyme in a 20 µl reaction volume are incubated for 30 minutes at 60°C. Reaction products are resolved by 20% denaturing polyacrylamide gel electrophoresis. Bands are cut out and radioactivity counted to quantify newly synthesized products.

STORAGE BUFFER: 20 mM Tris-HCl (pH 7.8), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, and 50% (v/v) glycerol.

STORAGE: Store at –20°C in a manual defrost freezer. For long-term storage, freeze at –80°C in working aliquots. Avoid repeated freeze-thawing. Enzyme may be diluted in 1X RECTM Buffer 15 for immediate use.

REFERENCE: Boudsocq, F, Iwai, S, Hanaoka, F, and Woodgate, R. 2001. Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic polη. Nucleic Acids Res. 29, 4607–4616. ORDERING INFORMATION

4150-010-EB Dpo4 polymerase, 10 µg, 10X REC™

Buffer 15, 1ml, 100 mM MgCl2, 1 ml,

and 50 mM MnCl2, 1 ml 4150-050-EB Dpo4 polymerase, 50 µg, 10X REC™

Buffer 15, 1ml, 100 mM MgCl2, 1 ml,

and 50 mM MnCl2, 1 ml

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DNA DAMAGE AND GENOMIC INSTABILITY

DNA Repair Enzymes Ku 70/80

Human Ku 70/80 Complex

CONTENTS: Catalog # Size 4135-250-01 250 Units

Human Ku 70/80 complex consists of 69.7 kDa and 82.6 kDa subunits, both of which act independently as ATP-dependent DNA helicases. The complex binds at double stranded DNA breaks and recruits DNA-PK, RAD50-Mre11-Xrs2, and XRCC4/DNA ligase IV to the site to facilitate nonhomologous end joining (NHEJ). The Ku 70/80 complex also participates in NHEJ during V(D)J recombination of immunoglobulin genes, which is necessary for generating the large antibody repertoire. The complex also restores activity to an immuno-depleted cell free DNA repair assay.

SOURCE: OVERVIEW Recombinant Ku heterodimer.

UNIT DEFINITION: 70/80 Ku One Unit is the amount of Ku 70/80 complex required to shift 100 fmoles of a labeled oligonucleotide duplex in 30 minutes at 25°C in an electrophoretic mobility shift assay.

SPECIFICITY: Ku 70/80 complex binds to ends of double stranded DNA breaks. 0065 ASSAY CONDITIONS: 1 pmole of an end labeled 24-mer oligonucleotide was incubated with 2 pmoles of a

complementary oligonucleotide in 25 mM Tris-Cl (pH 7.8), 0.5 mM EDTA, 5 mM MgCl2, 10 % glycerol, and 0.1% bromophenol blue in a reaction volume of 10 µl for 10 minutes at 65°C, and slow cooled to 22°C. Serial dilutions of the Ku 70/80 complex were incubated with the oligonucleotide duplex at 25°C for 30 minutes. Products were resolved on a 5% non-denaturing polyacrylamide gel.

STORAGE BUFFER: 50 mM Tris-Cl (pH 7.9), 0.3 M KCl, 1 mM EDTA, 0.02 % Tween 20, 15% glycerol, 1 mM DTT, and 10 µg/ml PMSF.

STORAGE: Store in working aliquots at -80˚C. Avoid repeated freeze thaws.

REFERENCES: 1. Dynan, W. and S. Yoo. 1998. Interaction of Ku protein and DNA dependent protein kinase catalytic subunit with nucleic acids. Nucleic Acids Res 26:1551-1559. 2. Yoo, S. and W. Dynan. 1999. Geometry of a complex formed by double stranded break repair proteins at a single DNA end: recruitment of DNA PKcs induces inward translocation of Ku protein. Nucleic Acids Res 27:4679-4686. 3. Hanakahi, L.A., and S.C. West. 2002. Specific interaction of IP6 with human Ku70/80, the DNA-binding subunit of DNA-PK. EMBO J. 21:2038-2044.

ORDERING INFORMATION

4135-250-01 Ku 70/80 Complex, 250 Units

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DNA DAMAGE AND GENOMIC INSTABILITY

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DNA Damage and Genomic Instability CANCER CELL BEHAVIOR

CANCER CELL BEHAVIOR

Apoptosis Detection

Stem Cell Products

Oxidative Stress

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CANCER CELL BEHAVIOR

Overview

To support the investigation of cell behavior in vitro and in vivo, Trevigen offers its Cultrex® line of products. It includes a full range of basement membrane extract (BME) and component proteins including Laminin 1 and Collagen IV. Also offered are Vitronectin, Fibronectin, Collagen 1 and assay kits designed to facilitate research in cellular adhesion, differentiation, migration, invasion, vascular permeability, angiogenesis, and tumorigenicity. Select Cultrex® products can be used for differentiation in 3-D culture environments and stem cell work. Trevigen’s assay kits are complete with matrix, staining reagents, controls, and plates. Our kits offer you the flexibility to optimize conditions with specific cell lines. Since the inception of the Cultrex® line, Trevigen’s scientists and internationally recognized consultants have worked hard to develop standardized manufacturing protocols to assure the delivery of consistent, high quality products to our customers. We initiated state of the art quality, sterility, infectious agent testing, and rigorous endotoxin specifications that are the most stringent in our industry. This resulted in our PathClear® BME. Finally our scientists have developed data regarding the use of our products that is available to you in the form of “data posters” and (or) peer reviewed publications. (see page 195). We recognize that in the future your research needs will continue to evolve and become more sophisticated. To help you meet your needs we will continue to develop new product applications, more rigorous manufacturing processes and more stringent performance specifications.

Please check our website for the latest product additions and product information.

Non transgenic primary mammary cells grown in Cultrex® 3-D Culture Matrix develop into a polarized acinus. Confocal The cancer cell behavior section is divided into five areas: CANCER CELL BEHAVIOR OVERVIEW CANCER CELL BEHAVIOR microscopy (5 µm projection) demonstrates epithelial polarity: DAPI stain, blue: GM130, red (Golgi protein, • Basement Membrane Extract (BME) 68 apical marker; panel A), Z01, green (tight junctions, • Extracellular Matrix (ECM) Component Proteins and Reagents apical; panel B); Integrin a6, magenta (baso-lateral; panel C), overlay shown in panel D. Images courtesy of • Cancer Cell Assays Martin Jechlinger. • 3-D Products • Cell Proliferation and Viability

HIGHLIGHTS: • Exclusive, Quantitative Directed In Vivo Angiogenesis Assay (DIVAA). • Pioneered unique Cell Invasion Optimization Assay System. • Instituted PCR, MAP and RAP testing for more than 30 infectious agents on both: starting biological materials and final products. • Lowered Endotoxin specification from 20 EU to 8 EU on BME.

DATA POSTERS (AVAILABLE UPON REQUEST): • Factors Affecting Basement Membrane Protein Dependent In Vitro Cell Models. 1. Tube Assay. 2. Cell Invasion Assay. 3. 3-D Culture Assays (see page 195).

REFERENCES: 1. Benton, G., and J. George. 2008. Using 3-D culture for high-throughput analysis of cytotoxicity and proliferation. Am Biotechnol Lab 26:24-7. 2. Benton G., E. Crooke, and J. George. 2009. Laminin-1 induces E-cadherin expression in 3-dimensional cultured breast cancer cells by inhibiting DNA methyltransferase 1 and reversing promoter methylation status. FASEB J. 23:3884-95. 3. Benton G, J. George, H.K. Kleinman, I.P. Arnaoutova. 2009. Advancing science and technology via 3-D culture on basement membrane matrix. J Cell Physiol. 221:18-25. 4. Arnaoutova I, J. George, H.K. Kleinman, G. Benton. 2009. The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. Angiogenesis. 12:267-74. 5. Arnaoutova I, and H.K. Kleinman. 2010. In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nat Protoc. 5:628-35. 6. Arnaoutova H, Benton G, George J, Kleinman H. Factors Affecting Basement Membrane Protein Dependent in vitro Cell Models. Tube Formation Assays Poster. Trevigen, Inc. 2009. 7. Arnaoutova H, Benton G, George J, Kleinman H. Factors Affecting Basement Membrane Protein Dependent in vitro Cell Models. Cell Invasion Assays Poster. Trevigen, Inc. 2009. 8. Arnaoutova H, Benton G, George J, Kleinman H. Factors Affecting Basement Membrane Protein Dependent in vitro Cell Models. 3-D Culture Assays Poster. Trevigen, Inc. 2009. 1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:16 PM Page 73

CANCER CELL BEHAVIOR

Cultrex® Product Selection Guide CULTREX ® 3-D CultureCell InvasionTube and Formation MigrationAdhesion Stem and AttachmentCellStem Propagation CellClonogenic Differentiation Assay AngiogenesisSubcutaneous AssayTumorograft “Plug”Product Assayand Xenograft Assays Description Catalog # Page

Vascular Permeability PRODUCT SELECTION GUIDE BME with and w/o phenol red 3430-005-01, 3432-005-01 70 BME with and w/o phenol red, PathClear 3430-005-02, 3432-005-02 72 BME Reduced Growth Factors, with and w/o phenol red 3431-005-01, 3433-005-01 70 BME Reduced Growth Factors, with and 3431-005-02, 3433-005-02 72 w/o phenol red, PathClear BME HC20+, PathClear 3444-005-02 74 OVERVIEW Human BME, PathClear 3415-001-02 80 Application BME Reduced Growth Factors, Phenol Red Free 3434-005-02 75 Stem Cell Qualified, PathClear 3-D Culture Matrix™ BME 3445-005-01 76 3-D Culture Matrix™ Laminin I 3446-005-01 106 3-D Culture Matrix™ Collagen I (Rat Tail) 3447-020-01 107 3-D Culture Cell Proliferation Assay-Core Kit 3445-096-CK 108 3-D Culture BME Cell Proliferation Assay Kit 3445-096-K 108 3-D Culture Laminin I Cell Proliferation Assay Kit 3446-096-K 108 3-D Culture Collagen I Cell Proliferation Assay Kit 3447-096-K 108 3-D Culture Cell Harvesting Kit 3448-020-K 109 0069 Laminin I (Mouse) 3400-010-01 83,84 Collagen IV (Mouse) 3410-010-01 86 Collagen I (Rat) 3440-100-01 85 Collagen I (Bovine) 3442-050-01 87 Fibronectin (Bovine) 3416-001-01 88 Fibronectin (Human), PathClear 3420-001-01 81 Vitronectin (Bovine) 3417-001-01 89 Vitronectin (Human), PathClear 3421-001-01 82 Vitronectin (Human), Reduced Nucleic Acids, PathClear 3422-001-01 82 Poly-L-Lysine 3438-100-01 91 Poly-D-Lysine 3439-100-01 90 In Vitro Angiogenesis Assay - CAS Tube Formation Kit 3470-096-K 97 In Vitro Angiogenesis Assay - Endothelial Cell Invasion Kit 3471-096-K 98 96 Well and 24 Well BME Cell Invasion Assay 3455-096-K, 3455-024-K 100 96 Well and 24 Well Laminin I Cell Invasion Assay 3456-096-K, 3456-024-K 100 96 Well and 24 Well Collagen I Cell Invasion Assay 3457-096-K, 3457-024-K 100 96 Well and 24 Well Collagen IV Cell Invasion Assay 3458-096-K, 3458-024-K 100 CultreCoat® 24 Well Cell Invasion Assay 3480-024-K 100 96 Well and 24 Well Cell Migration Assay 3465-096-K, 3465-024-K 100 96 Well and 24 Well In Vitro Vascular Permeability Kit 3475-096-K, 3475-024-K 99 CultreCoat® BME 96 Well Cell Adhesion Assay 3490-096-K 102 CultreCoat® Laminin I 96 Well Cell Adhesion Assay 3491-096-K 102 CultreCoat® Collagen I 96 Well Cell Adhesion Assay 3492-096-K 102 CultreCoat® Collagen IV 96 Well Cell Adhesion Assay 3493-096-K 102 CultreCoat® Fibronectin 96 Well Cell Adhesion Assay 3494-096-K 102 CultreCoat® Vitronectin 96 Well Cell Adhesion Assay 3495-096-K 102 CultreCoat® Adhesion Protein Array Kit 3496-096-K 102 Directed In Vivo Angiogenesis Assay (DIVAA) Inhibition Kit 3450-048-IK 95 Directed In Vivo Angiogenesis Assay Activation (DIVAA) Kit 3450-048-K 95 Directed In Vivo Angiogenesis Assay (DIVAA) Starter Kit 3450-048-SK 95 CultreCoat® 96 and 24 Well BME Cell Invasion 3484-096-K, 3484-024-K 100 Optimization Assay (Sampler) CultreCoat® 96 and 24 Well Low BME Cell Invasion Assay 3481-096-K, 3481-024-K 100 CultreCoat® 96 and 24 Well Medium BME Cell Invasion Assay 3482-096-K, 3482-024-K 100 CultreCoat® 96 and 24 Well High BME Cell Invasion Assay 3483-096-K, 3483-024-K 100

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Basement Membrane Extract (BME), Mouse

Basement membranes are continuous sheets of specialized extracellular matrix that form an interface between endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma. Basement membranes are degraded and regenerated during development and wound repair. They not only support cells and cell layers, but they also play an essential role in tissue organization that affects cell adhesion, migration, proliferation, and differentiation. Basement membranes provide major barriers to invasion by metastatic tumor cells.

Cultrex® Basement Membrane Extract (BME) is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor. The extract gels at 37°C to form a reconstituted basement membrane. The major components of BME include laminin, collagen IV, entactin, and heparin sulfate proteoglycan. BME is commonly used to maintain stem and precursor cells in an undifferentiated state during cell culture and BME hydrogels function to induce differentiation of epithelial, endothelial and smooth muscle cells in vitro. BME has also been employed in cell attachment, neurite outgrowth, angiogenesis, and in vitro cell invasion assays. BME is available with and without Phenol Red. Cultrex® BME is also available in PathClear® and HC20+™ varieties.

The PathClear® designation for EHS derived material means that in addition to standard sterility, endotoxin and MAP testing, the BME is tested by PCR and is clear of 31 pathogens and viruses, including lactate dehydrogenase elevating virus (LDEV), which replicates in macrophages. Each lot is rigorously qualified in biological performance assays. PathClear® BME is available

BME Mouse in normal and reduced growth factor form, with and without phenol red, and is ideal for in vivo murine research work and other work requiring BME free from viruses, bacteria and 70 mycoplasma.

Cultrex® BME PathClear® HC20+™ was developed for use in in vivo applications such as tumorigenicity assays, where higher protein concentrations facilitate faster gelling times, ORDERING INFORMATION increased gel strength, and elevated levels of tumor augmentation. Many primary tumor cells 3430-50-06 that will not proliferate in vitro, will proliferate as a xenograft when embedded in Cultrex® Phenol Red Solution, 50 µl BME PathClear® HC20+™.

For Cultrex® BME, PathClear®, see page 72

® TM ORDERING INFORMATION Cultrex 3-D Culture Matrix BME is prequalified for its ability to support the culture of cells in three dimensions. 3D culture is an innovative approach to modeling the morphological effects 3430-001-01 of early oncogenesis on glandular epithelial cells. When healthy, these cells exhibit a structured, Cultrex® BME with phenol red, 1 ml polarized morphology that is critical for tissue architecture and function. During carcinoma 3430-005-01 development, cell cycle controls associated with cellular development, proliferation and death Cultrex® BME with phenol red, 5 ml are lost, and as a result, acinar structure formation is disrupted. In effect, the morphology of 3431-001-01 these structures can be used as a measure to study factors in early carcinoma development. Cultrex® BME with phenol red, Reduced Growth Factor, 1 ml PHENOL RED: • Phenol red (catalog # 3430-50-01), provided separately at 100X needs to be added 3431-005-01 at 1:100 (v/v) prior to use. Cultrex® BME with phenol red, Reduced Growth Factor, 5 ml SHARED SPECIFICATIONS: 3432-001-01 • Endotoxin (LPS) Concentration: ≤ 8 EU/ml ® Cultrex BME, without phenol red, 1 ml • Source: Murine Engelbreth-Holm-Swarm (EHS) tumor 3432-005-01 • Storage Buffer: Dulbecco’s Modified Eagle’s medium containing 10 µg/ml gentamycin Cultrex® BME, without phenol red, 5 ml sulfate +/- phenol red. 3433-001-01 • Storage/Stability: Product is stable for a minimum of 3 months from date of shipment Cultrex® BME, without phenol red, when stored at –20 °C in a manual defrost freezer. For optimal stability, store at –80 °C Reduced Growth Factor, 1ml in aliquots. Keep Frozen; repeated freeze-thaws will destroy product integrity. 3433-005-01 Cultrex® BME, without phenol red, Reduced Growth Factor, 5 ml

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CANCER CELL BEHAVIOR

Basement Membrane Extract (BME), Mouse

FUNCTIONAL ASSAYS: • Tube Formation Assay: Basement Membrane Extract promotes formation of capillary-like structures by human (HUVEC, HMVEC) or mouse (SVEC4-10) endothelial cells.

• 3-D Culture: Basement Membrane Extract promotes differentiation of a human epithelial cell line derived from mammary gland (MCF-10A) and human prostate (PC-3) into acinar structures.

STANDARD STERILITY TESTING: • No bacterial or fungal growth detected after incubation at 37 °C for 14 days following USP XXIV Chapter 71 sterility test. • No mycoplasma contamination detected by PCR. • Endotoxin concentrations ≤ 8 EU/ml by LAL assay.

PATHCLEAR® STERILITY TESTING: OVERVIEW (Mouse) • Endotoxin concentrations ≤ 8 EU/ml by LAL assay. BME Mouse • No bacterial or fungal growth detected after incubation at 37 °C for 14 days following USP XXIV Chapter 71 sterility test. • No mycoplasma contamination detected by PCR. • Negative by PCR test for 17 bacterial and virus strains typically included in mouse antibody production (MAP) testing. These include lactate dehydrogenase elevating virus (LDEV) and 13 additional murine infectious agents, for a total of 31 organisms and viruses. 0071

Summary of available Basement Membrane Extract (BME) products

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CANCER CELL BEHAVIOR

Basement Membrane Extract (BME) Cultrex® Basement Membrane Extract, PathClear®

Cultrex® Basement Membrane Extract (BME) is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor. The extract gels at 37°C to form a reconstituted basement membrane. The major components of BME include laminin, collagen IV, entactin, and heparin sulfate proteoglycan. BME can be used to culture stem cells in the absence of feeder layers, and 3D hydrogels provide supports for organotypic cultures. BME has also been employed in cell attachment, neurite outgrowth, angiogenesis, in vitro cell invasion and in vivo tumorigenicity assays.

The PathClear® designation means that in addition to standard sterility, endotoxin and MAP testing, the BME is tested by PCR and is clear of 31 pathogens and viruses, including LDEV. Each lot is rigorously qualified in biological performance assays. PathClear® BME is available SVEC4-10 endothelial cells stained using Trevigen’s in normal and reduced growth factor form, with and without phenol red, and is ideal for ® Cultrex Cell Staining Kit (catalog # 3437-100-K). in vivo murine research work and other work requiring BME free from viruses, bacteria and mycoplasma. ® Specifications:

CONCENTRATION: 12–18 mg/ml.

SOURCE: BME PathClear Murine Engelbreth-Holm-Swarm (EHS) tumor.

72 STORAGE BUFFER: Dulbecco’s Modified Eagle’s medium containing 10 µg/ml gentamycin sulfate.

STORAGE/STABILITY: Product is stable for a minimum of 3 months from date of shipment when stored at –20°C in ORDERING INFORMATION a manual defrost freezer. For optimal stability, store at –80 °C in aliquots. Avoid repeated freeze/thaw cycles. 3430-001-02 Cultrex® BME with phenol red, PathClear® Material Qualification: (3432-001-02 & 3430-50-01), 1 ml 3430-005-02 FUNCTIONAL ASSAY: Cultrex® BME with phenol red, PathClear® • Tube Formation Assay: BME promotes formation of capillary-like structures by (3432-005-02 & 3430-50-01), 5 ml human (HUVEC, HMVEC) or mouse (SVEC4-10) endothelial cells. 3431-001-02 Cultrex® BME with phenol red, Reduced STERILITY TESTING: Growth Factor, PathClear® (3433-001-02 • No bacterial or fungal growth detected after incubation at 37 °C for 14 days following & 3430-50-01), 1 ml USP XXIV Chapter 71 sterility test. 3431-005-02 • Negative by PCR test for: mycoplasma, 17 bacterial and virus strains typically included in Cultrex® BME with phenol red, Reduced mouse antibody production (MAP) testing, plus 13 additional murine infectious agents Growth Factor, PathClear® (3433-005-02 including LDEV, for a total of 31 organisms and viruses. & 3430-50-01), 5 ml • Endotoxin concentrations ≤ 8 EU/ml by LAL assay. 3432-001-02 Cultrex® BME, without phenol red, PathClear®, 1 ml 3432-005-02 Cultrex® BME, without phenol red, PathClear®, 5 ml 3433-001-02 Cultrex® BME, without phenol red, Reduced Growth Factor, PathClear®, 1ml 3433-005-02 Cultrex® BME, without phenol red, Reduced Growth Factor, PathClear®, 5 ml

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CANCER CELL BEHAVIOR

Basement Membrane Extract (BME) Cultrex® Basement Membrane Extract, PathClear®

REFERENCES: 1. Albini, A., Y. Iwamoto, H. Kleinman, G. Martin, S. Aaronson, J. Kozlowski, and R. McEwan. 1987. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 47:3239-3245. 2. Fridman, R., G. Giaccone, T. Kanemoto, G. Martin, A. Gazdar, and J. Mulshine. 1990. Reconstituted basement membrane (matrigel) and laminin can enhance the tumorigenicity and the drug resistance of small cell lung cancer cell lines. Proc. Natl. Acad. Sci. USA 87:6698-6702. 3. Fridman, R., M. Kibbey, L.. Royce, M. Zain, T. Sweeney, D. Jicha, J. Yannelli, G. Martin, and H. Kleinman. 1991.Enhanced tumor growth of both primary and established human and murine tumor cells in athymic mice after coinjection with matrigel. J. Natl. Cancer Inst. 83:769-774. 4. Fridman, R., T. Sweeney, M. Zain, G. Martin, and H. Kleinman. 1992. Malignant transformation of NIH-3T3 cells after ubcutaneous co-injection with a reconstituted OVERVIEW basement membrane (matrigel). Int. J. Cancer 51:740-744. BME PathClear 5. Kubota, Y., H. Kleinman, G. Martin, and T. Lawley. 1988. Role of laminin and basement membrane proteins in the orphological differentiation of human endothelial cells in capillary-like structures. J. Cell Biol. 107:1589-1598. 6. Ponce, M., M. Nomizu, M. Delgado, Y. Kuratomi, M. Hoffman, S. Powell, Y. Yamada, H. Kleinman, and K. Malinda.1999. Identification of endothelial cell binding sites on the

laminin g1 chain. Circ. Res. 84:688-694. ® 7. Xu RH, Peck RM, Li DS, Feng X, Ludwig T and Thompson JA. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES. Nature Methods 2:185-190, 2005. 0073 8. Salcedo, R., H. Young, M. Ponce, J. Ward, H. Kleinman, J. Murphy, and J. Oppenheim. 2001. Eotaxin (CCL11) induces in vivo angiogenic responses by human CCR3+ endothelial cells. J. Immunol. 166:7571-7578. 9. Eisenstein, M. 2006. Thinking outside the dish. Nature Methods 3:1035-1043. 10. Benton, G., J. George, H.K. Kleinman, and I.P. Arnaoutova. 2009. Advancing Science and Technology Via 3D Culture on Basement Membrane Matrix. J. Cell. Physiol. 221:18-25. 11. Arnaoutova, I., J. George, H.K. Kleinman, and G. Benton. 2009. The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. Angiogenesis.12(3):267-74. 12. Arnaoutova I, and Kleinman HK. 2010. In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nat Protoc. 5(4):628-35.

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CANCER CELL BEHAVIOR

Basement Membrane Extract (BME) Cultrex® High Protein Concentration Basement Membrane Extract (HC20+™) PathClear®

Cultrex® BME HC20+™ PathClear® was developed for use in in vivo applications where higher protein concentrations facilitate faster gelling times, increased gel strength, and elevated levels of tumor augmentation. Cultrex® BME PathClear® HC20+™ has been tested by PCR and is clear of 31 pathogens and viruses, including LDEV, making it ideal for in vivo work. Cultrex® BME HC20+™ has the advantage of lot-to-lot consistency and controlled protein concentrations to support in vivo angiogenesis assays and tumorigenicity assays. Specifications: CONCENTRATION: ® ≥ 20 mg/ml

ENDOTOXIN (LPS) CONCENTRATION: ≤ 8 EU/ml

SOURCE: Murine Engelbreth-Holm-Swarm (EHS) tumor.

STORAGE BUFFER: Dulbecco’s Modified Eagle’s medium containing 10 µg/ml gentamycin sulfate HC20+™ BME, PathClear STORAGE/STABILITY: 74 Product is stable for a minimum of 3 months from date of shipment when stored at –20°C in a manual defrost freezer. For optimal stability, store at –80 °C in aliquots. Avoid repeated freeze/thaw cycles.

Material Qualification:

FUNCTIONAL ASSAY: • Tube Formation Assay: BME promotes formation of capillary-like structures by human (HUVEC, HMVEC) or mouse (SVEC4-10) endothelial cells.

STERILITY TESTING: • No bacterial or fungal growth detected after incubation at 37°C for 14 days following USP XXIV Chapter 71 sterility test. • Negative by PCR test for: mycoplasma, 17 bacterial and virus strains typically included in mouse antibody production (MAP) testing, plus 13 additional murine infectious agents including LDEV, for a total of 31 organisms and viruses.

REFERENCES: 1. Kubota, Y., H.K. Kleinman, G.R. Martin, and T.J. Lawley. 1988. Role of laminin and basement membrane proteins in the morphological differentiation of human endothelial cells into capillary-like structures. J. Cell. Biol. 107:1589-1598. 2. Ponce, M.L., M. Nomizu, M.C. Delgado, Y. Kuratomi, M.P. Hoffman, S. Powell, Y. Yamada, H.K. Kleinman, and K.M. Malinda. 1999. Identification of endothelial cell binding sites on the laminin γ1 chain. Circ. Res. 84:688-694. 3. Mirochnik, Y., A. Aurora, F.T. Schulze-Hoepfner, A. Deabes, V. Shifrin, R. Beckmann, C. Polsky, and O.V. Volpert. 2009. Short pigment epithelial-derived factor-derived peptide inhibits angiogenesis and tumor growth. Clin. Cancer Res. 15:1655–1663. 4. Guedez, L., A.M. Rivera, R. Salloum, M.L.Miller, J. J. Diegmueller, P.M. Bungay, and W.G. Stetler-Stevenson. 2003, Quantitative assessment of angiogenic responses by the ORDERING INFORMATION difrected in vivo angiogenesis assay. Am. J. Path. 162:1431-1439. 5. Mazibrada J, M. De Andrea M, Rittà M, Borgogna C, Dell'eva R, Pfeffer U, Chiusa L, 3444-005-02 Cultrex® High Protein Concentration Gariglio M, Landolfo S. 2010. In vivo growth inhibition of head and neck squamous BME (HC20+™), PathClear® 5 ml cell carcinoma by the Interferon-inducible gene IFI16. Cancer Lett. 287:33-43. 6. Fridman, R., M.C. Kibbey, L.S. Royce, M. Zain, T.M. Sweeney, D.L. Jicha, J.R. Yannelli, G.R. Martin, and H.K. Kleinman. 1991. Enhanced tumor growth of both primary and established human and murine tumor cells in athymic mice after coinjection with 1-800-873-8443 • www.trevigen.com Matrigel™. J. Natl. Cancer Inst. 83:769-774. INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:16 PM Page 79

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Basement Membrane Extract (BME) Cultrex® Stem Cell Qualified Basement Membrane Extract (BME), PathClear® A Cultrex® Stem Cell Qualified Basement Membrane Extract (BME), PathClear® is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor. The extract gels at 37 °C to form a reconstituted basement membrane. It is mainly comprised of laminin, collagen IV, entactin, and heparin sulfate proteoglycan. Cultrex® Stem Cell Qualified BME has STEM CELL QUALIFIED BME, PathClear been shown to provide an effective feeder-free surface for the attachment of and maintenance of human embryonic stem cells in a pluripotent state, thereby enabling its use for growth promotion or for study of stem cell differentiation.

B Specifications:

OVERVIEW CONCENTRATION:

12–18 mg/ml

SOURCE:

Murine Engelbreth-Holm-Swarm (EHS) tumor. ®

STORAGE BUFFER: 0075 C Dulbecco’s Modified Eagle’s medium containing 10 µg/ml gentamycin sulfate, without phenol red.

STORAGE/STABILITY:

Product is stable for a minimum of 3 months from date of shipment when stored at –20 °C in a manual defrost freezer. For optimal stability, store at –80 °C in aliquots; repeated freeze–thaws will destroy product integrity.

Material Qualification:

FUNCTIONAL ASSAY: H9 human embryonic stem cells after four passages on Cultrex® Stem Cell Qualified BME bind DAPI (A) and maintain expression of the non-differentiated stem cell • Promotes the attachment of H9 human embryonic stem cells. markers Oct-4 (B) and Nanog (C). • Effectively maintains human embryonic stem cells in a pluripotent state as evidenced by Images courtesy of the Yanik lab, MIT intracellular stains for the stem cell markers Oct-4 and Nanog. (www.rle.mit.edu/bbng) STERILITY TESTING:

• No bacterial or fungal growth detected after incubation at 37 °C for 14 days following USP XXIV Chapter 71 sterility test.

• Negative by PCR test for: mycoplasma, 17 bacterial and virus strains typically included in ORDERING INFORMATION mouse antibody production (MAP) testing, plus 13 additional murine infectious agents including LDEV, for a total of 31 organisms and viruses. 3434-001-02 ® Cultrex Stem Cell Qualified BME, • Endotoxin concentrations ≤ 8 EU/ml by LAL assay Reduced Growth Factor, PathClear®, 1 ml 3434-005-02 REFERENCES: Cultrex® Stem Cell Qualified BME, ® Reduced Growth Factor, PathClear , 5 ml 1. Angel, M. and M. F. Yanik. 2010. Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins. PLoS ONE. 5(7):e11756. 1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:16 PM Page 80

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Basement Membrane Extract (BME) Cultrex® 3-D Culture Matrix™ BME

3-D Culture Matrix™ reduced growth factor (RGF) BME provides the foundation for cells to grow in three dimensions, allowing for the formation of acinar and other hollow structures in vitro. Trevigen’s 3-D Culture Matrix™ products enable investigators to employ an innovative in vitro approach to modeling cell responses in in vivo-like environments, which cannot be observed in standard 2-D cultures1-3.

Specifications:

CONCENTRATION: 12–18 mg/ml

SOURCE: Murine Engelbreth-Holm-Swarm (EHS) tumor

STORAGE BUFFER: Dulbecco’s Modified Eagle’s medium containing 10 µg/ml gentamicin sulfate and no phenol red.

STORAGE/STABILITY: ® Non transgenic primary mammary cells grown in Cultrex 3-D Culture Product is stable for a minimum of 3 months from date of shipment when stored at -20°C in a Matrix develop into a polarized acinus. Confocal microscopy (5 µm projection) demonstrates epithelial polarity: DAPI stain, blue: manual defrost freezer. For optimal stability, store at -80 °C in aliquots. Avoid repeated GM130, red (Golgi protein, apical marker; panel A), Z01, green (tight freeze/thaw cycles. 3-D CULTURE MATRIX™ BME MATRIX™ 3-D CULTURE junctions, apical; panel B); Integrin a6, magenta (baso-lateral; panel C), overlay shown in panel D. Images courtesy of Martin Jechlinger. Material Qualification: 76 FUNCTIONAL ASSAY: • Acinar structure formation: 3-D Culture Matrix™ BME promotes differentiation of a human epithelial cell line derived from mammary gland (MCF-10A) into acinar structures. • Tube Formation Assay: BME promotes formation of capillary-like structures by human (HUVEC, HMVEC) or mouse (SVEC4-10) endothelial cells.

ORDERING INFORMATION

3445-001-01 Cultrex® 3-D Culture Matrix™ BME, 1 ml 3445-005-01 Cultrex® 3-D Culture Matrix™ BME, 5 ml

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ECM Component Proteins and Reagents Overview

The extracellular matrix (ECM) is the non-cellular environment outside of the cell that directs multicellular organization and provides structural support for tissues. The ECM provides anchorage for cells and connects directly to the cell cytoskeleton through transmembrane receptors. These interactions control vital cell functions, such as proliferation, differentiation, migration, polarity, and survival, and these processes are regulated through modulation of the cell’s epigenetic program and signal transduction cascades.

ECM proteins have revolutionized in vitro and in vivo cell models by providing optimal ECM COMPONENTS OVERVIEW environmental conditions to promote physiologically relevant cellular structure and function. These proteins have been used to: • Develop several organotypic models using 3-D culture • To provide barriers • To evaluate metastatic potential • To improve cellular implantation and evaluate angiogenesis in vivo • To maintain stem cells in an undifferentiated state OVERVIEW • To induce stem cell differentiation Trevigen proudly offers a full range of ECM proteins to meet your research needs.

Summary of Available Extracellular Matrix Component Proteins and Reagents:

0077

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ECM Component Proteins and Reagents

STANDARD STERILITY TESTING: (All ECM components) • No bacterial or fungal growth detected after incubation at 37°C for 14 days following USP XXIV Chapter 71 sterility test. • No Mycoplasma contamination detected by PCR. • Endotoxin concentrations ≤ 20 EU/ml by LAL assay.

PATHCLEAR® STERILITY TESTING: (Human BME) • Endotoxin concentrations ≤ 8 EU/ml by LAL assay. • No bacterial or fungal growth detected after incubation at 37°C for 14 days following USP XXIV Chapter 71 sterility test. • No Mycoplasma contamination detected by PCR. • Negative by PCR test for: eight human pathogenic viruses including Hepatitis A, B and C, MCF10A cells grown in collagen I gel , stained with Hoechst HIV 1 and 2, Hantaan, Seoul, and Sin Nombre. and anti-b-catenin antibody to visualize cell boundaries. Image courtesy of J. Partanen & J. Klefstrom, University PATHCLEAR® STERILITY TESTING: of Helsinki. Partanen, J.I., Makela, T.P. and (Other human proteins) Klefstrom, J. 2007. Suppression of oncogenic properties • Endotoxin concentrations ≤ 20 EU/ml by LAL assay. of c-Myc by LKB1-controlled epithelial organization. PNAS, 104: 14694 - 14699. • No bacterial or fungal growth detected after incubation at 37°C for 14 days following USP XXIV Chapter 71 sterility test. • No Mycoplasma contamination detected by PCR.

ECM COMPONENTS OVERVIEW • Negative by PCR test for: eight human pathogenic viruses including Hepatitis A, B and C, HIV 1 and 2, Hantaan, Seoul, and Sin Nombre. 78 PATHCLEAR® STERILITY TESTING: (Mouse proteins) • Endotoxin concentrations ≤ 8 EU/ml by LAL assay. • No bacterial or fungal growth detected after incubation at 37°C for 14 days following USP XXIV Chapter 71 sterility test. • No Mycoplasma contamination detected by PCR. • Negative by PCR test for 17 bacterial and virus strains typically included in mouse antibody production (MAP) testing including lactate dehydrogenase elevating virus (LDEV), plus 13 additional murine infectious agents, for a total of 31 organisms and viruses.

FUNCTIONAL ASSAYS: Fibronectin (Human & Bovine) The wells with the 20 µg/ml Cultrex® Fibronectin sample must exhibit HT1080 cell attachment of equal to or greater than 40%, whereas uncoated wells must have less than 10% attachment to pass quality control.

Vitronectin (Human & Bovine) The wells with the 20 µg/ml Cultrex® Vitronectin sample must exhibit HT1080 cell attachment of equal to or greater than 25%, whereas uncoated wells must have less than 10% attachment to pass quality control.

Laminin I (standard & PathClear®) The wells with the 20 µg/ml Cultrex® Laminin I sample must exhibit MG63 cell attachment of equal to or greater than 50%, whereas uncoated wells must have less than 10% attachment to pass quality control.

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ECM Component Proteins and Reagents

FUNCTIONAL ASSAYS: Collagen IV The wells with the 20 µg/ml Cultrex® Collagen IV sample must exhibit cell attachment of equal to or greater than 25%, whereas uncoated wells must have less than 10% attachment to pass quality control.

Rat Collagen I Gelled Rat Collagen I should support a formation of network-like structures of SVEC4-10 cells compared

to the cells adherent to the uncoated plastic surface ECM COMPONENTS OVERVIEW only. The wells with the 20 µg/ml Cultrex® Collagen I sample must exhibit cell attachment of equal to or greater than 40%, whereas uncoated wells must have less than 10% attachment to pass quality control.

Bovine Collagen I The wells with the 20 µg/ml Cultrex® Collagen I

sample must exhibit cell attachment of equal to or OVERVIEW greater than 40%, whereas uncoated wells must have less than 10% attachment to pass quality control.

Poly-L-Lysine The wells with the 0.01% Cultrex Poly-L-Lysine sample must exhibit cell attachment of equal to or greater than 10%, whereas uncoated wells must have less than 5% attachment to pass QC.

Poly-D-Lysine The wells with the 0.01% Cultrex Poly-D-Lysine 0079 sample must exhibit cell attachment of equal to or greater than 10%, whereas uncoated wells must have less than 5% attachment to pass QC.

*NZHD = New Zealand Herd Derived - New Zealand was granted negligible BSE risk disease status at the 75th General Session of the World Organization for Animal Health (OIE) (http://www.oie.int/eng/info/en_statesb.htm), and GBR I status by the European Food Safety Authority (http://ec.europa.eu/food/fs/sc/ssc/out292_en.pdf). No cases of BSE have been reported in New Zealand since monitoring began in 1989 (http://www.oie.int/eng/info/en_esbcarte.htm).

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ECM Component Proteins and Reagents Cultrex® Human BME, PathClear®

Cultrex® Human Basement Membrane Extract (BME) is a soluble form of basement membrane purified from human placenta. BME can be used for promotion and maintenance of an undifferentiated phenotype, including stem cells, primary epithelial cells, endothelial cells and smooth muscle cells. It has been employed in cell attachment assays, neurite outgrowth assays, and tumor cell invasion assays.

Specifications:

CONCENTRATION: ® 1 mg/ml

SOURCE: Human placenta

STORAGE BUFFER: Dulbecco’s Modified Eagle’s medium containing 10 µg/ml gentamycin sulfate

STORAGE/STABILITY: Product is stable for a minimum of a year from date of shipment when stored frozen at -80°C. Avoid repeated freeze/thaw cycles.

HUMAN BME, PathClear Material Qualification:

80 FUNCTIONAL ASSAY: Promotes the attachment of MG63 human osteosarcoma cells.

PATHCLEAR® STERILITY TESTING: • Endotoxin concentrations ≤ 8 EU/ml by LAL assay. • No bacterial or fungal growth detected after incubation at 37 °C for 14 days following USP XXIV Chapter 71 sterility test. • No mycoplasma contamination detected by PCR. • Negative by PCR test for: nineteen human pathogenic viruses including Hepatitis A, B and C, HIV 1 and 2, Hantaan, Seoul, Sin Nombre, Human T-lymphotropic Virus 1 & 2, Epstein Barr, Herpes Simplex 1 & 2, Human Cytomegalovirus, Human Herpes Virus 6, Human Herpes Virus 8, Human Adenovirus, Varicella Virus, Lymphocytic choriomeningitis Virus.

SAFETY STATEMENT: Although Cultrex® Human BME PathClear® has been tested for the viruses above it contains human source material and therefore should be treated as potentially infectious and handled at the Biological Safety Level 2 to minimize exposure.

ORDERING INFORMATION

3415-001-02 Cultrex® Human BME PathClear®, 1 ml

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ECM Component Proteins and Reagents Cultrex® Human Fibronectin, PathClear®

Fibronectin is an extracellular matrix protein that is found abundantly in blood and connective tissues. These matrices are associated with the epithelial to mesenchymal transition of metastatic cells--including tumor cells with stem cell-like properties. Fibronectin performs essential functions in collagen fibrillogenesis, as either a general cell adhesion molecule or a modulator in binding between cell surfaces and the extracellular matrix. Fibronectin matrix assembly is essential for normal vertebrate development and apparently contributes to the

generation of tumor metastases by supporting the establishment and persistence of HUMAN FIBRONECTIN, PathClear pre-metastatic niches. Fibronectin is secreted as a disulfide-linked dimer of 230-270 kDa, comprised of three types of repeating modules that mediate interactions with extracellular matrix components (including fibronectin itself), and cells via integrins and other fibronectin receptors. Thus, fibronectin can be used for coating tissue culture surfaces or as a medium additive to promote cell adhesion and proliferation; it is 0.2 µm sterile filtered.

Specifications: OVERVIEW CONCENTRATION: 1 mg/ml

SOURCE: Human plasma ® STORAGE BUFFER: 100 mM CAPS, 150 mM NaCl, 1 mM CaCI2, pH 11.5 0081 STORAGE: Store at -80oC in a manual defrost freezer. Avoid freeze-thaw cycles.

PURITY: > 90% by SDS-PAGE gel

Material Qualification:

PATHCLEAR® STERILITY TESTING: • No bacterial or fungal growth detected after incubation at 37 °C for 14 days following USP XXIV Chapter 71 sterility test. • No mycoplasma contamination detected by PCR. • Human Fibronectin has tested negative for eight human pathogenic viruses including Hepatitis A, B and C, HIV 1 and 2, Hantaan, Seoul, and Sin Nombre by PCR. • Endotoxin concentrations ≤ 20 EU/ml by LAL assay.

FUNCTIONAL ASSAY: Tested for ability to promote attachment of HT1080 cells.

SAFETY STATEMENT: Although Cultrex® Human Fibronectin, PathClear® has been tested for the viruses above it contains human source material and therefore should be treated as potentially infectious and handled at the Biological Safety Level 2 to minimize exposure.

REFERENCES: 1. Vaheri A, Mosher DF. (1978) High molecular weight, cell surface-associated glycoprotein (fibronectin) lost in malignant transformation. Biochim Biophys Acta. 516(1):1-25. 2. Mao Y, Schwarzbauer JE. (2005) Fibronectin fibrillogenesis, a cell-mediated matrix ORDERING INFORMATION assembly process. Matrix Biol 24:389-99. 3. Polyak K, Weinberg RA. (2009) Transitions between epithelial and mesenchymal states: 3420-001-01 Cultrex® Human Fibronectin, PathClear®, acquisition of malignant and stem cell traits. Nat Rev Cancer 9:265-73. 1 mg

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ECM Component Proteins and Reagents Cultrex® Human Vitronectin, PathClear®

Vitronectin is an extracellular, soluble, disulfide-linked dimer, composed of a 75 kDa and a 65 kDa peptide chain with a total molecular weight of 140 kDa. Vitronectin is a major plasma glycoprotein that promotes cellular adhesion and spreading. It also inhibits the membrane- damaging effect of the terminal cytolytic complement pathway and binds to several serpin serine protease inhibitors. Vitronectin, along with collagen IV, fibronectin, and laminin can support robust, long term proliferation of undifferentiated human embryonic stem cells.

® Vitronectin can be used for coating tissue culture surfaces to promote cell adhesion, proliferation and differentiation, or as an additive for serum-free media. Vitronectin is purified from human plasma and is 0.2 µm sterile filtered.

Cultrex® Human Vitronectin, PathClear® is also available in a Nuleic Acids Reduced form.

Specifications:

CONCENTRATION: 1 mg/ml

SOURCE: Human plasma

STORAGE BUFFER: HUMAN VITRONECTIN, PathClear 10 mM Sodium Phosphate, pH 7.7, 8M Urea, 5 mM EDTA, 500 mM NaCl

82 STORAGE: Store at -80°C in a manual defrost freezer. Avoid freeze-thaw cycles.

PURITY: > 90% by SDS-PAGE gel

Material Qualification:

FUNCTIONAL ASSAY: Tested for ability to promote attachment of HT1080 cells.

PATHCLEAR® STERILITY TESTING: • No bacterial or fungal growth detected after incubation at 37 °C for 14 days following USP XXIV Chapter 71 sterility test. • No mycoplasma contamination detected by PCR. • Human Vitronectin has tested negative for eight human pathogenic viruses including Hepatitis A, B and C, HIV 1 and 2, Hantaan, Seoul, and Sin Nombre by PCR. • Endotoxin concentrations ≤ 20 EU/ml by LAL assay.

SAFETY STATEMENT: Although Cultrex® Human Vitronectin, PathClear® has been tested for the viruses above it contains human source material and therefore should be treated as potentially infectious and handled at the Biological Safety Level 2 to minimize exposure.

REFERENCES: ORDERING INFORMATION 1. Vuento M, Korkolainen M, Kuusela P, Holtta E. (1985) Isolation of a novel cell-attachment and spreading-promoting protein from human serum. Biochem J. 227:421-7. 3421-001-01 2. Hayman EG, Pierschbacher MD, Suzuki S, Ruoslahti E (1985). Vitronectin--a major cell Cultrex® Human Vitronectin PathClear®, attachment promoting protein in fetal bovine serum. Exp Cell Res. 160:245-58. 50 µg 3. Tschopp J, Masson D, Schafer S, Peitsch M, Preissner KT (1988). The heparin binding 3422-001-01 domain of S protein/vitronectin binds to complement components C7, C8, and C9 and perforin Cultrex® Human Vitronectin, Nuleic Acid from cytolytic T cells and inhibits their lytic activities. Biochemistry 27:4103-9. Reduced, PathClear®, 50 µg

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ECM Component Proteins and Reagents Cultrex® Mouse Laminin I

Laminin I is an extracellular matrix protein which contains a number of functional domains that allow it to assemble into sheets. Trevigen’s mouse Laminin I is purified from the murine EHS sarcoma. This highly purified preparation increases cell adhesion, migration, proliferation, and differentiation.1 It is composed of α1β1γ1 chains with a total Mr of 800,000 and is used for coating culture dishes.

Embryonic stem (ES) cells express receptors for laminin. Thus, the use of laminin as a growth matrix should be considered when peptide growth factors present in more complex hydrogels might adversely impact experimental results. In pure form, laminin I is sufficient to support excellent ES cell growth in the undifferentiated state in the absence of feeder cells2.

Specifications:

CONCENTRATION: MOUSE LAMININ I 1 mg/ml OVERVIEW

SOURCE: Murine Engelbreth-Holm-Swarm (EHS) tumor

STORAGE BUFFER: Dulbecco’s Modified Eagle’s medium containing 10 µg/ml gentamycin sulfate.

STORAGE/STABILITY: Store at -20°C or -80°C in a manual defrost freezer. Avoid repeated freeze/thaw cycles. 0083

PURITY: > 90% by SDS-PAGE gel

Material Qualification:

FUNCTIONAL ASSAY: Supports the attachment (at least 50%) of MG63 human osteosarcoma cells to a laminin-coated plate at a coating solution concentration of 20 µg/ml. Model of 3-D Laminin-induced Bleomycin Resistance in Metastatic Breast Cancer. 3-D Cultured MDA-MB-231 cells STERILITY TESTING: are resistant to bleomycin. In the presence of PJ34, a PARP • No bacterial or fungal growth detected after incubation at 37 °C for 14 days following inhibitor, they become sensitive to bleomycin. USP XXIV Chapter 71 sterility test. • No mycoplasma contamination detected by PCR • Endotoxin concentrations ≤ 20 EU/ml by LAL assay.

REFERENCES: 1. Benton G., E. Crooke, and J. George. 2009. Laminin-1 induces E-cadherin expression in 3-dimensional cultured breast cancer cells by inhibiting DNA methyltransferase 1 and reversing promoter methylation status. FASEB J. 23:3884-95. 2. Xu, C., M.S Inokuma., J. Denham, K. Golds, P. Kundu, J.D. Gold, and M.K. Carpenter. 2001. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19:971–974.

ORDERING INFORMATION

3400-010-01 Cultrex® Mouse Laminin I, 1 mg

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ECM Component Proteins and Reagents Cultrex® Mouse Laminin I, PathClear®

This highly purified preparation of mouse laminin I also increases cell adhesion, migration, proliferation, and differentiation. It is also composed of α1β1γ1 chains with a total Mr of 800,000 and is used for coating culture dishes. The PathClear® designation means that in addition to standard sterility, endotoxin and MAP testing, the Laminin is tested by PCR and is clear of 31 pathogens and viruses, including lactate dehydrogenase elevating virus (LDEV) which proliferates in macrophages. Each lot is rigorously qualified in biological performance assays.

® Specifications:

CONCENTRATION: 120% 1 mg/ml 100% (%) 80% SOURCE: 60% Murine Engelbreth-Holm-Swarm (EHS) tumor

Adhesion 40% 20% STORAGE BUFFER: 0% Dulbecco’s Modified Eagle’s medium containing 10 µg/ml gentamycin sulfate. 0 5 10 20 Concentration(μg/ml) STORAGE/STABILITY: Store at -20°C or -80°C in a manual defrost freezer. Avoid repeated freeze/thaw cycles.

MOUSE LAMININ I, PathClear Laminin I promotes adhesion of MG63 cells. Laminin I was diluted to 5, 10, and 20 µg/ml and 100 µl of each solution was added to six wells each of a 96 well plate and incubated PURITY: 84 overnight at 37°C. The coating solution was then aspirated, and > 90% by SDS-PAGE gel wells were blocked for nonspecific binding with a 2% BSA solution. MG63 cells were labeled with calcein am, harvested, Material Qualification: and seeded at 15,000 cells/well. The cells were allowed to attach for one hour and 15 minutes. Total fluorescence was assessed in a 96 well plate reader. The cells were aspirated off and the FUNCTIONAL ASSAY: plate was washed with PBS before reading fluorescence again. Supports the attachment (at least 50%) of MG63 human osteosarcoma cells to a laminin-coated The percent adhesion was calculated from fluorescence of plate at a coating solution concentration of 20 µg/ml. adhered cells divided by the fluorescence of total cells loaded for each well. STERILITY TESTING: • No bacterial or fungal growth detected after incubation at 37 °C for 14 days following USP XXIV Chapter 71 sterility test. • PathClear®: Negative by PCR test for: mycoplasma, 17 bacterial and virus strains typically included in mouse antibody production (MAP) testing, plus 13 additional murine infectious agents including LDEV, for a total of 31 organisms and viruses. • Endotoxin concentrations ≤ 20 EU/ml by LAL assay.

ORDERING INFORMATION

3400-010-02 Cultrex® Mouse Laminin I, PathClear®, 1 mg

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ECM Component Proteins and Reagents Cultrex® Rat Collagen I

Type I collagen is the major structural component of extracellular matrices found in connective tissue and internal organs, but is most prevalent in the dermis, tendons, and bone. It is a 300 kDa molecule composed of two alpha1(I) chains and one alpha2(I) chain that spontaneously forms a triple helix scaffold at a neutral pH and 37°C. This phenomenon can be exploited to promote cell attachment, proliferation, differentiation, migration, and tissue morphogenesis during development.

Specifications:

CONCENTRATION: 5 mg/ml (Sircol Assay)

SOURCE: Rat tail tendons RAT COLLAGEN I OVERVIEW STORAGE BUFFER: 20 mM Acetic Acid, pH 3.4 - 3.6

STORAGE/STABILITY: Product is stable for a minimum of 3 months from date of shipment if stored at 4°C. Do Not Freeze.

MCF10A cells grown in collagen I gel and stained with PURITY: Hoechst and anti-b-catenin antibody to visualize cell boundaries. Image courtesy of J. Partanen & J. Klefstrom, > 90% by SDS-PAGE gel 0085 University of Helsinki. Partanen, J.I., Makela, T.P. and Klefstrom, J. 2007. Suppression of oncogenic properties Material Qualification: of c-Myc by LKB1-controlled epithelial organization. PNAS, 104: 14694 - 14699. FUNCTIONAL ASSAY: Tested for the ability to promote cell attachment and spreading of HT1080 human fibrosarcoma cells.

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ECM Component Proteins and Reagents Cultrex® Mouse Collagen IV

Collagen IV is a primary collagen found in the basement membranes, which underlie epithelial and endothelial cells and surround muscle, fat and nerve cells. Collagen IV can be used for coating of tissue culture surfaces to promote cell attachment and proliferation and to study its effects on cell behavior.

Specifications:

CONCENTRATION: 0.5 mg/ml

SOURCE: Murine Engelbreth-Holm-Swarm (EHS) tumor

STORAGE BUFFER: 50 mM Hydrochloric Acid, 1 mM TCEP - HCl

STORAGE/STABILITY: Store at -80°C in a manual defrost freezer if using the product within 3 months. Avoid repeated freeze-thaw cycles. For optimal stability, store in liquid nitrogen.

PURITY: MOUSE COLLAGEN IV > 90% by SDS-PAGE gel

86 Material Qualification:

FUNCTIONAL ASSAY: Tested for ability to promote attachment and spreading of rat PC-12 pheochromocytoma cells.

REFERENCES: 1. Hudson B.G., S.T. Reeders , and Tryggvason K. 1993. Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis. J. Biol. Chem. 268, 26033-36.

ORDERING INFORMATION

3410-010-01 Cultrex® Mouse Collagen IV, 1 mg

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ECM Component Proteins and Reagents Cultrex® Bovine Collagen I

Specifications:

CONCENTRATION: 5 mg/ml (Sircol Assay) BOVINE COLLAGEN I SOURCE:

Fetal Bovine Extensor Tendons OVERVIEW

STORAGE BUFFER: 20 mM Acetic Acid, pH 3.4 - 3.6

STORAGE/STABILITY: Product is stable for a minimum of 3 months from date of shipment if stored at 4°C. Do Not Freeze.

Material Qualification: 0087 FUNCTIONAL ASSAY: Tested for the ability to promote cell attachment and spreading of HT1080 human fibrosarcoma cells.

REFERENCES: 1. Chen, S., R. Revoltella, S. Papini, M. Michelini, W. Fitzgerald, J. Zimmerberg, and L. Margolis. 2003. Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems. Stem Cells. 21:281-295. 2. Kokenyesi, R., K. Murray, A. Benshushan, E. Huntley, and M. Kao.2003. Invasion of interstitial matrix by a novel cell line from primary peritoneal carcinosarcoma, and by established ovarian carcinoma cell lines: role of cell-matrix adhesion molecules, proteinases and E-cadherin expression. Gynecol Oncol. 89:60-72. 3. Kutznetsova, N., S. Chi, and S. Leikin. 1998. Sugars and polyols inhibit fibrillogenesis of type I collagen by disrupting hydrogen-bonded water bridges between the helices. Biochem. 37:11888-11895. 4. Kutznetsova, N., and S. Leikin. 1999. Does the triple helical domain of type I collagen encode molecular recognition and fiber assembly while telopeptides serve as catalytic domains. J. Bio. Chem. 274:36083-36088. 5. Leikin, S., D. Rau, and V. Parsegian. 1994. Direct measurement of forces between self-assembled proteins: Temperature-dependent exponential forces between ORDERING INFORMATION collagen triple helices. Proc. Natl. Acad. Sci. USA. 91:276-280. 6. Leikina, E., M. Mertts, N. Kuznetsova, and S. Leikin. 2002. Type I collagen is thermally unstable 3442-005-01 at body temperature. Proc. Natl. Acad. Sci. USA. 99:1314-1318. Cultrex Collagen I (Bovine), 5 mg 7. O’ Shaughnessy, T., H. Lin, and W. Ma. 2003. Functional synapse formation among rat cortical 3442-050-01 neurons grown on three-dimensional collagen gels. Neuroscience Letters. 340:169 - 172. Cultrex Collagen I (Bovine), 50 mg 8. Park, D., D. Choi, H. Ryu, H. Kwon, H. Joo, and C. Min. 2003. A well-defined in vitro three-dimensional culture of human endometrium and its applicability to endometrial cancer invasion. Cancer Letters. 195:185-192. 1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:17 PM Page 92

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ECM Component Proteins and Reagents Cultrex® Bovine Fibronectin NZHD*

Fibronectin is an extracellular, soluble, disulfide-linked dimer composed of two 230-270 kDa independent globular peptide chains with a total molecular weight of 440 kDa. Fibronectin is an extracellular matrix protein that is found abundantly in blood, connective tissues, and provisional matrices associated with malignant transformation of metastatic cells. Fibronectin functions either as a general cell adhesion molecule or as a modulator in binding between cell surfaces and the extracellular matrix by means of a central cell binding domain, RGD (Arg-Gly-Asp). Fibronectin can be used for coating tissue culture surfaces to promote cell adhesion or as a medium additive.

Specifications:

CONCENTRATION: 1 mg/ml

SOURCE: Bovine plasma

STORAGE BUFFER: 5X Phosphate Buffered Saline

STORAGE/STABILITY:

BOVINE FIBRONECTIN NZHD* Store at -20°C or -80°C in a manual defrost freezer. Avoid freeze-thaw cycles.

88 PURITY: > 90% by SDS-PAGE gel

Material Qualification:

FUNCTIONAL ASSAY: Tested for ability to promote attachment of HT1080 cells.

REFERENCES: 1. Vaheri A, Mosher DF. High molecular weight, cell surface-associated glycoprotein (fibronectin) lost in malignant transformation. Biochim Biophys Acta. 1978 Sep 18; 516(1):1-25. 2. Edelman GM. Cell adhesion molecules, Science. 1983 Feb 4; 219(4584):450-7.

ORDERING INFORMATION 3416-001-01 Cultrex® Bovine Fibronectin, 1 mg

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ECM Component Proteins and Reagents Cultrex® Bovine Vitronectin NZHD*

Vitronectin is an extracellular, soluble, disulfide-linked dimer, composed of a 75 kDa and a 65 kDa peptide chain with a total molecular weight of 140 kDa. Vitronectin is a major plasma glycoprotein that promotes cellular adhesion and spreading, inhibits the membrane-damaging effect of the terminal cytolytic complement pathway, and binds to several serpin serine protease inhibitors. Vitronectin can be used for coating tissue culture surfaces to promote cell adhesion, proliferation and differentiation, or as an additive for serum-free media.

Specifications: BOVINE VITRONECTIN NZHD* SOURCE: Bovine plasma

STORAGE BUFFER: 10 mM Sodium Phosphate, pH 7.7, 8M Urea, 5 mM EDTA, 500 mM NaCl. OVERVIEW STORAGE/STABILITY: Store at -80°C in a manual defrost freezer. Avoid freeze-thaw cycles.

PURITY: > 90% by SDS-PAGE gel

Material Qualification:

FUNCTIONAL ASSAY: 0089 Tested for ability to promote attachment of HT1080 cells.

ORDERING INFORMATION

3417-001-01 Cultrex® Bovine Vitronectin, 50 µg

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ECM Component Proteins and Reagents Cultrex® Poly-D-Lysine

Poly-D-Lysine, a highly positively charged synthetic amino acid chain, is commonly used as a coating agent to promote cell adhesion in culture on TCT plastic or glass surfaces. Moreover, poly-D-Lysine is resistant to enzymatic degradation and promotes the proliferation and differentiation of a variety of neuronal cell lines. This solution is provided ready to use at 0.01% and contains polymers in the 70,000-150,000 kDa range.

APPLICATIONS: Substrate for cell culture adhesion. An area of 25 cm2 can be coated with 0.5 ml of a 0.01% Poly-D-Lysine solution. Optimal conditions for attachment must be determined for each cell line and application. Slides may be dipped in the solution and air dried before applying sample.

CONCENTRATION: 0.01% in phosphate-buffered saline (PBS), sterile-filtered.

STORAGE: Product is stable for at least 6 months from the date of receipt when stored at 2 - 8°C. Keep sterile.

Specifications:

POLY-D-LYSINE FUNCTIONAL ASSAY: Tested for ability to promote attachment of rat PC-12 pheochromocytoma cells. 90 STERILITY TESTING: • No bacterial or fungal growth detected after incubation at 37oC for 14 days following USP Chapter 71 sterility testing. • Endotoxin concentration ≤ 20 EU/mL by LAL assay.

MYCOPLASMA TESTING: Not required for this synthetic product.

REFERENCES: 1. Tsuyuki E, Tsuyuki H, Stahmann MA. (1956) The synthesis and enzymatic hydrolysis of poly-D-lysine. J Biol Chem. 222:479-85. 2. Tombran-Tink J, Johnson LV. (1989) Neuronal differentiation of retinoblastoma cells induced by medium conditioned by human RPE cells. Invest Ophthalmol Vis Sci. 30:1700-7. 3. Hayashi Y, Furue MK, Okamoto T, Ohnuma K, Myoishi Y, Fukuhara Y, Abe T, Sato JD, Hata R, Asashima M. (2007) Integrins Regulate Mouse Embryonicm Stem Cell Self-Renewal. Stem Cells 25:3005-15.

ORDERING INFORMATION 3439-100-01 Cultrex Poly-D-Lysine, 100 ml

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ECM Component Proteins and Reagents Cultrex® Poly-L-Lysine

This highly positively charged amino acid chain is commonly used as a coating agent to promote cell adhesion in culture. This solution is provided ready to use at 0.01% and contains polymers in the 70,000–150,000 kDa range.

CONCENTRATION: 0.01% in Phosphate Buffered Saline, sterile-filtered.

VOLUME: 100 ml

STORAGE CONDITIONS: Store at 4˚C. Keep sterile.

SOURCE: OVERVIEW

Fermentation using starch POLY-L-LYSINE

APPLICATIONS: Substrate for cell culture adhesion. An area of 25 cm2 can be coated with 0.5 ml of a 0.01% Poly-L-Lysine solution. Optimal conditions for attachment must be determined for each cell line and application. Slides may be dipped in the solution and air dried before applying sample.

CONCENTRATION: 0.01% in phosphate-buffered saline (PBS), sterile-filtered. 0091 STORAGE: Product is stable for at least 6 months from the date of receipt when stored at 2 - 8°C. Keep sterile. Attachment and spreading of PC-12 cells rat adrenal gland pheochromocytoma grown on a plate coated with 0.01% Poly-L-Lysine. QUALITY CONTROL: • Tested for ability to promote attachment of rat PC-12 pheochromocytoma cells. • Cell Culture Qualified: No bacterial or fungal growth detected after incubation at 37˚C for 14 days following USP XXIV, Chapter 71 sterility testing. No mycoplasma contamination detected by PCR. • Endotoxin concentration ≤ 20 EU/ml by LAL assay.

REFERENCES: 1. Harper JM, Huynh M, Coppens I, Parussini F, Moreno S, Carruthers VB. (2006) A cleavable propeptide influences toxoplasma infection by facilitating the trafficking and secretion of the TgMIC2-M2AP invasion complex. Mol. Biol. Cell 17:4551-4563.

ORDERING INFORMATION

3438-100-01 Cultrex® Poly-L-Lysine, 100 ml

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ECM Component Proteins and Reagents 3-D Culture Matrix™ Mouse Laminin I

3-D Culture Matrix™ Mouse Laminin I may be used as a gel on which to grow cells, or as a media additive alone or in concert with other basement membrane components, to study cellular growth and differentiation in three dimensions in vitro. To offer the most standardized Laminin I for use in 3-D cultures, a special process is employed to provide material at a standard concentration of 6 mg/ml (by absorbance and extinction coefficient).

Specifications:

CONCENTRATION: 6 mg/ml MOUSE LAMININ I

TM PURITY: ≥90% (SDS-PAGE)

SOURCE: Murine Engelbreth-Holm-Swarm (EHS) tumor

STORAGE BUFFER: Dulbecco’s Modified Eagle’s medium containing 10 µg/ml gentamycin sulfate and no phenol red.

STORAGE/STABILITY: 3-D CULTURE MATRIX 3-D CULTURE Product is stable for a minimum of 3 months from date of shipment when stored at -80 °C in a manual defrost freezer. For optimal stability, store at -80 °C in aliquots. 92 Avoid repeated freeze/thaw cycles.

Material Qualification:

FUNCTIONAL ASSAY: • 3-D Culture: Laminin I promotes differentiation of a human epithelial cell line derived from mammary gland (MCF-10A) into acinar structures. • Cell Attachment: Tested for the ability to promote cell attachment and spreading of MG63 human osteosarcoma cells.

STERILITY TESTING: • No bacterial or fungal growth detected after incubation at 37°C for 14 days following USP XXIV Chapter 71 sterility test. • No mycoplasma contamination detected by PCR. Primary embryonic submandibular epithelium cultured in • Endotoxin concentrations ≤ 20 EU/ml by LAL assay. laminin-1 gel with FGF10 (200 ng/ml). The Epithelia are cultured for either 24 or 48 hours in serum-free media and undergo branching morphogenesis in culture. Image courtesy of Matthew P. Hoffman. Patel, V.N., Knox, S.M., Likar, K.M., Lathrop, C.A., Hossain, R., Eftekhari, S., Elkins, M., Vlodasky, I., Whitelock, J.M. and Hoffman, M.P. Heparanase cleavage of heparin sulfate modulates FGF10 function during submandibular gland branching morphogenesis. Development, 2007, 134(23):4177-86.

ORDERING INFORMATION

3446-005-01 Cultrex® 3-D Culture Matrix™ Mouse Laminin I, 30 mg

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ECM Component Proteins and Reagents 3-D Culture Matrix™ Rat Collagen I

The 3-D Culture Matrix™ Rat Collagen I may be used as a gel on which to grow cells or a medium additive, alone or in concert with other basement membrane components, to study cellular

growth and differentiation in three dimensions in vitro. Type I Collagen is the major structural 3-D CULTURE MATRIX™ RAT COLLAGEN I component of extracellular matrices found in connective tissue and internal organs, but is most prevalent in the dermis, tendons, and bone. It is a 300 kDa molecule composed of two alpha1(I) chains and one alpha2(I) chain that spontaneously forms a triple helix scaffold. This phenomenon can be exploited to promote cell attachment, growth, differentiation, migration, and tissue morphogenesis during development.

Specifications:

CONCENTRATION: 5 mg/ml (Sircol Assay) OVERVIEW SOURCE: Rat tail tendons

STORAGE BUFFER: 20 mM Acetic Acid, pH 3.4 - 3.6

STORAGE/STABILITY: Product is stable for a minimum of 3 months from date of shipment if stored at 4°C. Do Not Freeze. 0093 Material Qualification:

FUNCTIONAL ASSAY: • 3-D Culture: Collagen I forms a gel when diluted to 0.4 mg/ml at neutral pH and promotes attachment and growth of murine endothelial SVEC4-10 cells. • Cell Attachment: Tested for the ability to promote cell attachment and spreading of HT1080 human fibrosarcoma cells.

REFERENCES: 1. Benton, G., and J. George. 2008. Using 3-D culture for high-throughput analysis of cytotoxicity and proliferation. Am Biotechnol Lab 26:24-27. 2. Benton G., Crooke E., and George J. 2009. Laminin-1 induces E-cadherin expression in 3-dimensional cultured breast cancer cells by inhibiting DNA methyltransferase 1 and reversing promoter methylation status. FASEB J. 23:3884-95. 3. Benton G, George J, Kleinman HK, Arnaoutova IP. 2009. Advancing science and technology via 3-D culture on basement membrane matrix. J Cell Physiol. 221:18-25.

ORDERING INFORMATION

3447-020-01 Cultrex® 3-D Matrix™ Rat Collagen I, 100 mg

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Cancer Cell Assays Overview

To support the investigation of cell behavior in vitro and in vivo, Trevigen scientists have developed, invented or improved upon our Cultrex® line of cell assay kits. The kits are designed to facilitate research in cellular adhesion, differentiation, migration, invasion, vascular permeability, angiogenesis, tumorigenicity, as well as differentiation in 3D culture environments and stem cell work. Trevigen’s assay kits are complete with instructions for use, BME or purified matrix proteins, staining reagents, controls, and plates. Our kits offer you high-throughput multi-well formats, or sufficient flexibility in a 24 well format to optimize assay conditions for your cell lines. We back our kits with in-house quality control, and expert technical support. Moreover, Trevigen scientists have published protocols, interesting findings, and technical insights in peer-reviewed scientific journals.1,2

Summary of Available Cancer Cell Assay Kits:

Storage/ Max Stability Catalog # Product Application For Components Size List Price Page

3450‐048‐SK Cultrex® DIVAA Starter Kit In Vivo Angiogenesis ‐20°C, 4°C and RT 48 Wells 95 3450‐048‐K Cultrex® DIVAA Activation Kit In Vivo Angiogenesis ‐20°C, 4°C and RT 48 Wells 95 3450‐048‐IK Cultrex® DIVAA Inhibition In Vivo Angiogenesis ‐20°C, 4°C and RT 48 Wells 95 3470‐096‐K Cultrex® In Vitro Angiogenesis Assay Tube Formation Kit In Vitro Angiogenesis ‐20°C and RT 96 Wells 97 3471‐096‐K Cultrex® In Vitro Angiogenesis Assay Endothelial Cell Invasion Kit Endothelial Cell Angiogenesis ‐20°C and RT 96 Wells 98 3475‐024‐K CultreCoat® 24 Well In Vitro Vascular Permeability Assay Vascular Permeability 4°C 24 Wells 99

CANCER CELL ASSAYS OVERVIEW CANCER CELL ASSAYS 3475‐096‐K CultreCoat® 96 Well In Vitro Vascular Permeability Assay Vascular Permeability 4°C 96 Wells 99 3480‐024‐K CultreCoat® 24 Well BME Cell Invasion Assay Cell Invasion ‐20°C, 4°C and RT 24 Wells 100 3484‐096‐K CultreCoat® 96 Well BME Cell Invasion Optimization Assay Cell Invasion Optimization ‐20°C, 4°C and RT 96 Wells 100 94 3484‐024‐K CultreCoat® 24 Well BME Cell Invasion Optimization Assay Cell Invasion Optimization ‐20°C, 4°C and RT 24 Wells 100 3483‐096‐K CultreCoat® 96 Well High BME Cell Invasion Assay Cell Invasion Optimization ‐20°C, 4°C and RT 96 Wells 100 3483‐024‐K CultreCoat® 24 Well High BME Cell Invasion Assay Cell Invasion Optimization ‐20°C, 4°C and RT 24 Wells 100 3482‐096‐K CultreCoat® 96 Well Medium BME Cell Invasion Assay Cell Invasion Optimization ‐20°C, 4°C and RT 96 Wells 100 3482‐024‐K CultreCoat® 24 Well Medium BME Cell Invasion Assay Cell Invasion Optimization ‐20°C, 4°C and RT 24 Wells 100 3481‐096‐K CultreCoat® 96 Well Low BME Cell Invasion Assay Cell Invasion Optimization ‐20°C, 4°C and RT 96 Wells 100 3481‐024‐K CultreCoat® 24 Well Low BME Cell Invasion Assay Cell Invasion Optimization ‐20°C, 4°C and RT 24 Wells 100 3465‐096‐K Cultrex® 96 Well Cell Migration Assay Cell Migration ‐20°C, 4°C and RT 96 Wells 100 3465‐024‐K Cultrex® 24 Well Cell Migration Assay Cell Migration ‐20°C, 4°C and RT 24 Wells 100 3455‐096‐K Cultrex® 96 Well BME Cell Invasion Assay Cell Invasion ‐20°C, 4°C and RT 96 Wells 100 3455‐024‐K Cultrex® 24 Well BME Cell Invasion Assay Cell Invasion ‐20°C, 4°C and RT 24 Wells 100 3456‐096‐K Cultrex® 96 Well Laminin I Cell Invasion Assay Cell Invasion ‐20°C, 4°C and RT 96 Wells 100 3456‐024‐K Cultrex® 24 Well Laminin I Cell Invasion Assay Cell Invasion ‐20°C, 4°C and RT 24 Wells 100 3457‐096‐K Cultrex® 96 Well Collagen I Cell Invasion Assay Cell Invasion ‐20°C, 4°C and RT 96 Wells 100 3457‐024‐K Cultrex® 24 Well Collagen I Cell Invasion Assay Cell Invasion ‐20°C, 4°C and RT 24 Wells 100 3458‐096‐K Cultrex® 96 Well Collagen IV Cell Invasion Assay Cell Invasion ‐20°C, 4°C and RT 96 Wells 100 3458‐024‐K Cultrex® 24 Well Collagen IV Cell Invasion Assay Cell Invasion ‐20°C, 4°C and RT 24 Wells 100 3496‐096‐K CultreCoat® Adhesion Protein Array Kit Cell Adhesion ‐20°C, 4°C and RT 96 Wells 102 3490‐096‐K CultreCoat® BME 96 Well Cell Adhesion Assay Cell Adhesion ‐20°C, 4°C and RT 96 Wells 102 3491‐096‐K CultreCoat® Laminin I 96 Well Cell Adhesion Assay Cell Adhesion ‐20°C, 4°C and RT 96 Wells 102 3492‐096‐K CultreCoat® Collagen I 96 Well Cell Adhesion Assay Cell Adhesion ‐20°C, 4°C and RT 96 Wells 102 3493‐096‐K CultreCoat® Collagen IV 96 Well Cell Adhesion Assay Cell Adhesion ‐20°C, 4°C and RT 96 Wells 102 3494‐096‐K CultreCoat® Fibronectin 96 Well Cell Adhesion Assay Cell Adhesion ‐20°C, 4°C and RT 96 Wells 102 3495‐096‐K CultreCoat® Vitronectin 96 Well Cell Adhesion Assay Cell Adhesion ‐20°C, 4°C and RT 96 Wells 102 3445‐096‐CK Cultrex® 3D Culture BME Cell Proliferation Assay Core Kit Cell Proliferation ‐20°C, 4°C and RT 96 Wells 108 3445‐096‐K Cultrex® 3D Culture BME Cell Proliferation Assay Kit Cell Proliferation ‐20°C, 4°C and RT 96 Wells 108 3446‐096‐K Cultrex® 3D Culture Laminin I Cell Proliferation Assay Kit Cell Proliferation ‐20°C, 4°C and RT 96 Wells 108 3447‐096‐K Cultrex® 3D Culture Collagen I Cell Proliferation Assay Kit Cell Proliferation ‐20°C, 4°C and RT 96 Wells 108 4890‐25‐K TACS® MTT Cell Proliferation Assay Cell Proliferation 4°C and RT 2500 Wells 111 4890‐50‐K TACS® MTT Cell Proliferation Assay Cell Proliferation 4°C and RT 5000 Wells 111 4891‐025‐K TACS® XTT Cell Proliferation Assay Cell Proliferation ‐20°C 2500 Wells 111 4892‐010‐K Cultrex® Calcein‐AM Cell Viability Kit Cell Viability ‐20°C and 4°C 1000 Wells 111 3448‐020‐K Cultrex® 3‐D Culture Cell Harvesting Kit 3‐D Culture ‐20°C, 4°C and RT 20 Wells 109 3437‐100‐K Cultrex® Cell Staining Kit Cell Staining RT 100 ml 104

REFERENCES: 1. Arnaoutova, I., J. George, H.K. Kleinman, and G. Benton. 2009. The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. Angiogenesis. 12:267-74. 2. Arnaoutova I, and Kleinman HK. 2010. In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nat Protoc. 5:628-35. 1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:18 PM Page 99

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Cancer Cell Assays Cultrex® Directed In Vivo Angiogenesis Assay (DIVAATM)

DIVAA is the first in vivo system for the study of angiogenesis that provides quantitative and reproducible results.1 During the course of the assay, implant grade silicone cylinders closed at one end, called angioreactors, are filled with 20 µl of Trevigen's PathClear® basement membrane extract (BME) premixed with or without angiogenic-modulating factors*. These angioreactors are then implanted subcutaneously in the dorsal flank of nude mice. Accompanied with the onset of angiogenesis, vascular endothelial cells proceed to grow into the BME and form vessels in the angioreactor. As early as nine days post-implantation, there are enough cells to determine an effective dose response to angiogenic modulating factors. The sleek design of the angioreactor provides a standardized platform for reproducible and quantifiable in vivo angiogenesis assays. Compared to the plug assay5, the angioreactor prevents assay errors due to absorption of the basement membrane extract by the mouse. In addition, the angioreactor Angioreactor filled with RGF BME (with phenol red for color uses only a fraction of the materials conserving both BME and test compounds used, and up contrast). to four angioreactors may be implanted in each mouse, allowing for greater statistical power. Trevigen’s DIVAA has been used in a variety of investigations.1-4, 6-9. OVERVIEW DIVAA is available in three formats:

DIVAA Starter Kit Catalog # 3450-048-SK DIVAA The DIVAA Starter Kit was designed to introduce the technology and give the user practical experience assessing angiogenesis. It contains 48 Angioreactors, enough growth factor to

induce angiogenesis all 48 angioreactors, and an Angiorack™ designed to hold the TM Angioreactors during the course of assay setup.

DIVAA Activation Kit Catalog # 3450-048-K 0095 The DIVAA Activation Kit was designed for assessing angiogenesis activation. It contains 48 Implantation of angioreactors. angioreactors and enough growth factor for eight positive controls.

DIVAA Inhibition Assay Catalog # 3450-048-IK The DIVAA Inhibition Kit was designed for assessing angiogenesis inhibition. It contains 48 Angioreactors and enough growth factor to induce angiogenesis in all 48 angio-reactors.

* The PathClear® designation means:

• No bacterial or fungal growth detected after incubation at 37°C for 14 days following USP XXIV Chapter 71 sterility test. • Negative by PCR test for: mycoplasma, 17 bacterial and virus strains typically included in mouse antibody production (MAP) testing, plus 13 additional murine infectious agents including LDEV, for a total of 31 organisms and viruses.

ORDERING INFORMATION

3450-048-SK 48 Wells, DIVAA™ Starter Kit 3450-048-IK 48 Wells, DIVAA™ Inhibition Kit 3450-048-K 48 Wells, DIVAA™ Activation Kit 3450-048-DA 48 Wells, DIVAA™ Angioreactors and BME (3450-048-01 & 3450-048-02) 3450-048-FL 48 Wells, DIVAA™ FITC-Lectin and Buffer (3450-048-06 & 3450-048-07)

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Cancer Cell Assays Cultrex® Directed In Vivo Angiogenesis Assay (DIVAATM)

REFERENCES: 1.Guedez L, Rivera AM, Salloum R, Miller ML, Diegmueller JJ, Bungay PM, Stetler-Stevenson WG. 2003. Quantitative Assessment of Angiogenic Response by the Directed In Vivo Angiogenesis Assay. AJP 162:1431-1439. 2. Seo D, Li H, Guedez L, Wingfield PT, Diaz T, Salloum R, Wei B, Stetler-Stevenson WG. 2003. TIMP-2 Mediated Inhibition of Angiogenesis: An MMP-Independent Mechanism. Cell 114:171-180. 3. Martinez A, Vos M, Guedez L, Kaur G, Chen Z, Garayoa M, Pio R, Moody T, Stetler- Stevenson WG, Kleinman HK, Cuttitta F. 2002. The effects of adrenomedullin overexpression in breast tumor cells. J Natl Cancer Inst 94:1226-37. 4. Wang T, Ward Y, Tian L, Lake R, Guedez L, Stetler-Stevenson WG, Kelly K. 2005. CD97, an adhesion receptor on inflammatory cells, stimulates angiogenesis through binding integrin counterreceptors on endothelial cells. Blood 105:2836-44. 5. Lee MS, Moon EJ, Lee SW, Kim MS, Kim KW, Kim YJ. 2001. Angiogenic activity of pyruvic acid in in vivo and in vitro angiogenesis models. Cancer Res 61:3290-3. 6. Kurozumi K, Hardcastle J, Thakur R, Shroll J, Nowicki M, Otsuki A, Chiocca EA, Kaur B. 2008. Oncolytic HSV-1 infection of tumors induces angiogenesis and upregulates CYR61. Mol Ther 16:1382-91. TM 7. Basile JR, Holmbeck K, Bugge TH, Gutkind JS. 2007. MT1-MMP controls tumor- induced angiogenesis through the release of semaphoring 4D. J Biol Chem 282:6899-905. 8. Liu S, Wang H, Currie BM, Molinolo A, Leung HJ, Moayeri M, Basile JR, Alfano RW, DIVAA Gutkind JS, Frankel AE, Bugge TH, Leppla SH. 2008. Matrix-metalloproteinase-activated anthrax lethal toxin demonstrates high potency in targeting tumor vasculature. J Biol 96 Chem 283:529-40. 9. Sakamoto K, Maeda S, Hikiba Y, Nakagawa H, Hayakawa Y, Shibata W, Yanai A, Ogura K, Omata M. 2009. Constitutive NF-κB activation in colorectal carcinoma plays a key role in angiogenesis, promoting tumor growth. Clin Cancer Res 15:2248-58. 10. U.S. Patent 4,829,000 11. U.S. Patent 5,158,874

This product is made and marketed under patent license from the United States Public Health Service. Ref. U.S. Patent 4,829,000 issued May 9, 1989 and U.S. Patent 5,158,874 issued October 27,1992, all entitled Reconstituted Membrane Complex with Biological Activity.

A B

Figure 7. Vascularization in DIVAA™ angioreactor. New vessel formation is appearant in the DIVAA™ RGF BME inside the angior actor prior to excission (A), and after harvest from the angioreactor (B). Photo Provided By William Stetler-Stevenson. 1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:18 PM Page 101

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Cancer Cell Assays Cultrex® In Vitro Angiogenesis Assay Tube Formation Kit

Under permissive conditions, endothelial cells respond by self organizing into structures that resemble microvessels or tubes. Trevigen's Cultrex® In Vitro Angiogenesis Assay Kit allows for the detection of inducers and inhibitors of endothelial cell tube formation. Cultrex® Reduced Growth Factor (RGF) Basement Membrane Extract (BME) is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor, which gels at room temperature to form a reconstituted protein matrix comprised mainly of laminin, collagen IV, entactin, and heparin sulfate proteoglycan. Sulforaphane [1- isothiocyanato-(4R)- methylsulfinyl)-butane], found in broccoli and other cruciferous vegetables, is a naturally occurring cancer chemopreventive agent, and is provided as a control for inhibition of in vitro endothelial cell tube formation on Cultrex® RGF BME. Calcein AM is provided for rapid and accurate measurement of cell viability and/or cytotoxicity, and kinetic analysis of tube formation. Trevigen's Cultrex® In Vitro Angiogenesis Assay Kit provides a cost-effective TUBE FORMATION KIT method for investigation of prospective angiogenesis inhibitors in a 96 well plate format.

REFERENCES: OVERVIEW 1. Folkman J. 2007. Angiogenesis: an organizing principle for drug discovery? Nat. Rev. Drug Discov. 6:273-286. 2. Jaffe EA, Nachman RL, Becker CG, Minick CR. 1973. Culture of human endothelial cells derived from umbilical veins. J. Clin. Invest. 52:2745-2756. 3. Folkman J, Haudenschild C. 1980. Angiogenesis in vitro. Nature 288:551- 556. 4. Kleinman HK, McGarvey ML, Hassell JR, Star VL, Cannon FB, Laurie GW, Martin GR. 1986. Basement membrane complexes with biological activity. Biochemistry 25:312-318. 5. Lawley TJ, Kubota Y. 1989. Induction to morphologic differentiation of endothelial cells in culture. J. Invest. Dermatol. 93:59S-61S. 0097 6. Ucuzian AA, Greisler HP. 2007. In vitro models of angiogenesis. World J. Surg. 31:654-663. 7. Salcedo R, Zhang X, Young HA, Michael N, Wasserman K, Ma W-H, Martins-Green M, Murphy WJ, Oppenheim JJ. 2003. Angiogenic effects of prostaglandin E2 are mediated by up-regulation of CXCR4 on human microvascular endothelial cells. Blood 102:1966-1977. 8. Mochizuki M, Philp D, Hozumi K, Suzuki N, Yamada Y, Kleinman HK, Nomizu M. 2007. Angiogenic activity of syndecan-binding laminin peptide AG73 (RKRLQVQLSIRT). Arch. Biochem. Biophys. 459:249-255. 9. Arnaoutova, I., J. George, H.K. Kleinman, and G. Benton. 2009. The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. Angiogensis. 12(3):267-74. 10. Arnaoutova I, and Kleinman HK. 2010. In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nat Protoc. 5(4):628-35.

ORDERING INFORMATION Human Umbilical Vein Endothelial Cells (HUVEC) cultured for six hours on gelled RGF BME in Endothelial Basal Medium (panel A), Endothelial Growth Medium (panel B) or in Endothelial Growth Medium in the presence of 15 µM Sulforaphane (panel C). 3470-096-K Cultrex® In Vitro Angiogenesis Assay Tube Formation Kit, 96 Wells

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Cancer Cell Assays Cultrex® In Vitro Angiogenesis Assay Endothelial Cell Invasion Kit

During angiogenesis, which is the formation of neovasculature associated with wound healing and a host of disease states including tumor growth, endothelial cells invade through the basement membrane to form sprouting vessels, which in turn invade hypoxic tissue. Trevigen's Cultrex® In Vitro Angiogenesis Assay Endothelial Cell Invasion Kit was created in an effort to accelerate the screening process for compounds that influence vascular endothelial cell digestion through a basement membrane extract (BME) layer. This kit offers a flexible, standardized, 96 well high-throughput format for quantitating endothelial invasion, in vitro, in response to chemoattractants and/or inhibiting compounds. This assay employs a simplified Boyden chamber-like design with an 8 micron polyethylene terephthalate (PET) membrane. Sulforaphane [1-isothiocyanato-(4R)-methylsulfinyl)-butane], a naturally occurring cancer chemopreventive agent, is provided as a control for inhibition of in vitro endothelial cell invasion through Cultrex® BME. Detection of cell invasion is quantified using Calcein-AM. Calcein AM is internalized by the cells, and intracellular esterases cleave the acetomethylester (AM) moiety. Free Calcein fluoresces brightly, and this fluorescence may be used to quantitate the number of cells that have invaded or migrated using a standard curve.

REFERENCES: 1. Hanahan D, Weinberg RA. (2000) The hallmarks of cancer. Cell 100:57–70. 2. Medina MA, Munoz-Chapuli R, Quesada AR. (2007) Challenges of antiangiogenic cancer therapy: trials and errors, and renewed hope. J Cell Mol Med 11:374-82. ENDOTHELIAL CELL INVASION KIT ENDOTHELIAL CELL INVASION 3. Qian X, Wang TN, Rothman VL, Nicosia RF, Tuszynski GP. (1997) Thrombospondin-1 modulates angiogenesis in vitro by up-regulation of matrix metalloproteinase-9 in 98 endothelial cells. Exp Cell Res 235:403–12. 4. Chantrain CF, Shimada H, Jodele S, Groshen S, Ye W, Shalinsky DR, Werb Z, Coussens LM, DeClerck YA. (2004) Stromal matrix metalloproteinase-9 regulates the vascular architecture in neuroblastoma by promoting pericyte recruitment.Cancer Res 64:1675-86. 5. Arnaoutova I, George J, Kleinman HK, Benton G. (2009) The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. Angiogenesis. 12:267-74. 6. Arnaoutova I, and H.K. Kleinman. (2010) In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nat Protoc. 5:628-35.

Figure: Relative quantification (as a percent of the migration control) of the ability of endothelial cell lines (SVEC4-10, HUVEC and HBMVEC) to traverse through 8 micron polyester filters coated with a 1X BME (Basement Membrane Extract) solution over a 24 hour period in response to VEGF, bFGF and other chemoattractants contained in Endothelial Growth Medium-2 (EGM-2), in the presence or absence of 5 µM ORDERING INFORMATION Sulforaphane. Endothelial Basal 3471-096-K Medium (EBM) Cultrex® In Vitro Angiogenesis Assay was used as a negative control due Endothelial Cell Invasion Kit, 96 Wells to absence of chemoattractants.

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Cancer Cell Assays CultreCoat® Vascular Permeability Assays

The vascular endothelium is the thin monolayer of cells that lines blood vessels and provides a network for the exchange of biological materials such as gases, nutrients, and metabolic waste throughout the body. The endothelium exhibits a selective barrier function between the vessel lumen and the surrounding tissues which controls this exchange. There are many vasoactive cytokines and growth factors that function in regulating the degree of vascular

permeability, such as interleukin-1 alpha and beta1, TNF-alpha2, IFN gamma1, hepatocyte growth VASCULAR PERMEABILITY ASSAYS factor (HGF)3, and vascular endothelial growth factor (VEGF)4. There are also several systemic diseases associated with disruption of vascular permeability, such as cancer4, diabetes3, heart disease3, stroke5, hypertension5, and arthritis6.

Trevigen’s Cultrex® Vascular Permeability Assays were created in an effort to accelerate the screening process for signal transduction pathway modulators and compounds that influence vascular permeability. These assays offers a flexible, standardized, high-throughput format for quantitating the degree to which genes or compounds can influence the maintenance of endothelial cell-to-cell adhesion. OVERVIEW

Figure 2 Cytochalasin B increases vascular permeability. HUVECs were These assays are available as either individual inserts in a 24 well plate or a 96 well chamber

seeded at 100,000 cells/well and cultured for 72 hours at 37°C in a CO2 to accommodate individual experiments or large scale screening. These assays employ a incubator. Cells were treated with Cytochalasin B for five hours and simplified Boyden chamber design with polyethylene terephthalate (PET) membrane coated ® evaluated for vascular permeability using the CultreCoat 96 Well with Collagen I. Additional ports within the migration chamber (top) allow access to the assay In Vitro Vascular Permeability Assay. chamber (bottom) without dismantling the device. This design is easier to use and helps to prevent microbial and cross contamination. The 96 well assay is adaptable for robotic high throughput systems, and the assay chamber may be directly analyzed in a 96 well plate reader, eliminating transfer steps that introduce additional variability. 0099

Detection of vascular permeability is quantified using FITC-Dextran. The confluent cell monolayer forms a barrier through cell-cell adhesion mechanisms such as junctions (adherens, tight, and gap) and desmosomes, and this barrier restricts access of the FITC-Dextran to the top chamber of the inserts. Disruption of these cell-cell adhesion mechanisms creates gaps in the intercellular space that allow diffusion of FITC-Dextran into the bottom chamber. The amount of FITC-Dextran that diffuses is related to the amount of intercellular space, which is representative of vascular permeability.

REFERENCES: 1. Martin, S.S., IL-1 and IFN-gamma increase vascular permeability. Immunology, 1988. 64:2:301. 2. Edamitsu, S., et al., Role of TNF[alpha], IL-1, and IL-1ra in the Mediation of Leukocyte Infiltration and Increased Vascular Permeability in Rabbits with LPS-Induced Pleurisy. Clinical Immunology and Immunopathology, 1995. 75:1:68-74. 3. Emani, S., et al., Increased vascular permeability after cardiopulmonary bypass in patients with diabetes is associated with increased expression of vascular endothelial growth factor and hepatocyte growth factor. The Journal of Thoracic and Cardiovascular Surgery, 2009. 138:1:185-191. 4. Cornali, E.E., Vascular endothelial growth factor regulates angiogenesis and vascular permeability in Kaposi's sarcoma. The American Journal of Pathology, 1996. 149(6): p. 1851. 5. Lee, J.-M., et al., Vascular Permeability Precedes Spontaneous Intracerebral Hemorrhage in Stroke-Prone Spontaneously Hypertensive Rats. Stroke, 2007. 38:12:3289-3291. 6. Huang, M., et al., Mast cell deficient W/W(v) mice lack stress-induced increase in serum ORDERING INFORMATION IL-6 levels, as well as in peripheral CRH and vascular permeability, a model of rheumatoid arthritis. International Journal of Immunopathology and Pharmacology, 2002. 15:3:249-254. 3475-024-K CultreCoat® 24 Well In Vitro Vascular Permeability Assay 3475-096-K CultreCoat® 96 Well In Vitro Vascular Permeability Assay

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Cancer Cell Assays ORDERING INFORMATION Cultrex® Cell Invasion and Migration Assays 3484-024-K ® CultreCoat 24 Well BME Cell Invasion The Cultrex® Cell Invasion Assays were created in an effort to accelerate the screening process Optimization Assay (sampler) for compounds that influence cellular digestion and migration across extracellular matrices, 3484-096-K 1 ® which is a fundamental component of cellular processes such as angiogenesis , embryonic CultreCoat 96 Well BME Cell Invasion 2 3 4 Optimization Assay (sampler) development , immune responses , and tumor cell metastasis . These assays offer a flexible, standardized, high-throughput format for quantitating the degree to which invasive cells penetrate a barrier consisting of basement membrane components in vitro in response to chemoattractants and/or inhibiting compounds.

3455-024-K These assays employ a simplified Boyden chamber design with a polyethylene terephthalate Cultrex® 24 Well BME Cell Invasion Assay (PET) membrane containing 8 micron pores, which allow access from the input chamber (top) 3455-096-K to the assay chamber (bottom) without dismantling the device. This design is easier to use, ® Cultrex 96 Well BME Cell Invasion Assay prevents contamination, and is adaptable for robotic high throughput systems. The assay 3456-024-K chamber may be directly analyzed in a 96 well plate reader, eliminating transfer steps that ® Cultrex 24 Well Laminin I Cell introduce additional variability to the assay. Invasion Assay 3456-096-K Cell invasion is quantified using calcein-acetoxymethyl ester (Calcein-AM), which is internalized ® Cultrex 96 Well Laminin I Cell by the cells, and cleaved by intracellular esterases to generate fluorescence signals. Invasion Assay In conjunction with a standard curve, these signals may be used to quantitate the number of 3457-024-K cells that have migrated or invaded—thereby eliminating the need for direct cell counting. These Cultrex® 24 Well Collagen I Cell Invasion Assay kits are available as either individual inserts within 24 well plates or a complete 96 well chambers, allowing researchers to choose a format that best fits their experimental design. 3457-096-K CELL INVASION AND MIGRATION ASSAYS AND MIGRATION CELL INVASION Cultrex® 96 Well Collagen I Cell Invasion Assay Cell Invasion and Migration may be evaluated using three formats: 100 3458-024-K Cultrex® 24 Well Collagen IV Cell CultreCoat® Cell Invasion Optimization System Invasion Assay

3458-096-K ® Cultrex® 96 Well Collagen IV Cell The CultreCoat Optimization System is a flexible approach that allows you to choose the Invasion Assay desired degree of cell invasion necessary for a specific project. Included in the system are a series of Boyden chambers coated at three distinct concentrations of BME and uncoated wells 3480-024-K CultreCoat® Original 24 Well BME Cell to compare invasion to basal migration. Relative to uncoated wells, cell invasion decreases as Invasion Assay the concentration of the BME coating increases. This system provides an easy approach to 3481-024-K determine the ideal coating density that provides the maximum signal-to-noise ratio and CultreCoat® 24 Well Low BME Cell dynamic required for your study. Once the desired concentration is determined, BME coated Invasion Optimization Assay plates can be ordered. The system is available in both 96 well and 24 well formats. 3481-096-K CultreCoat® 96 Well Low BME Cell ® Invasion Optimization Assay Cultrex Cell Invasion Assays 3482-024-K CultreCoat® 24 Well Medium BME Cell Cultrex® Cell Invasion Assays provide a system where the researcher can coat their plates Invasion Optimization Assay having the option to choose from several extracellular matrix proteins. Since different cell lines 3482-096-K and different treatments can result in a wide range of invasive potentials, the permissiveness CultreCoat® 96 Well Medium BME Cell of each matrix may also be optimized to fit each experiment by adjusting the coating Invasion Optimization Assay concentration. Cultrex® Cell Invasion Assays are provided in multiple formats for evaluation 3483-024-K against different extracellular matrices and matrix components: Laminin I, Collagen I, Collagen CultreCoat® 24 Well High BME Cell IV, and Basement Membrane Extract (BME) Invasion Optimization Assay 3483-096-K ® CultreCoat® 96 Well High BME Cell Cultrex Cell Migration Assays Invasion Optimization Assay The Cultrex® Cell Migration Assays provide uncoated Boyden chambers with all of the reagents necessary to assess cell migration in the absence of extracellular matrix proteins.

3465-024-K Cultrex® 24 Well Cell Migration Assay 3465-096-K Cultrex® 96 Well Cell Migration Assay

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Cancer Cell Assays Cultrex® Cell Invasion and Migration Assays

REFERENCES: 1. Tamilarasan KP, Kolluru GK, Rajaram M, Indhumathy M, Saranya R, Chatterjee S. 2006. Thalidomide attenuates nitric oxide mediated angio-genesis by blocking migration of endothelial cells. BMC Cell Biol. 7:17. CELL INVASION AND MIGRATION ASSAYS 2. Borghesani PR, Peyrin JM, Klein R, Rubin J, Carter AR, Schwartz PM, Luster A, Corfas G, Segal RA. 2002. BDNF stimulates migration of cerebellar granule cells. Development 129:1435-1442. 3. Mohan K, Ding Z, Hanly J, Issekutz TB. 2002 IFN-gamma-inducible T cell alpha chemoattractant is a potent stimulator of normal human blood T lymphocyte transendothelial migration: differential regulation by IFN-gamma and TNF-alpha. J Immunol. 168:6420-6428. 4. Li G, Chen YF, Greene GL, Oparil S, Thompson JA. 1999 Estrogen inhibits vascular smooth muscle cell-dependent adventitial fibroblast migration in vitro. Circulation 100:1639-1645. 5. Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, Kozlowski JM, McEwan RN. 1987. A rapid in vitro assay for quantitating the invasive potential of tumor cells.

Cancer Res. 47:3239-45. OVERVIEW

10100

Different coating densities for the 96 Well BME Cell Invasion Chamber results in different invasion rates for invasive cell lines. HT-1080, human fibrosarcoma, and MDA-MB-231, breast cancer, cell lines were serum starved for 16 hours and seeded at 25,000 cells per well in pre-coated 8 µm chambers. Cells invaded in response to 10% FBS over a 24 hour period and were quantitated using Calcein-AM. Samples were run in quadruplicate.

1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 11/31/112:18PMPage106 102 CELL ADHESION ASSAYS 1-800-873-8443 3496-096-K CultreCoat 3496-096-K CultreCoat 3495-096-K CultreCoat 3494-096-K CultreCoat 3493-096-K CultreCoat 3492-096-K CultreCoat 3491-096-K CultreCoat 3490-096-K Adhesion Assay Adhesion Assay Adhesion Assay Adhesion Assay Assay Adhesion Adhesion Assay REIGINFORMATION ORDERING ® ® ® ® ® ® ® Adhesion ProteinArrayKit Vitronectin 96Well Cell Fibronectin 96Well Cell Collagen IV96Well Cell Collagen I96Well Cell Laminin I96Well Cell BME 96Well Cell

• www.trevigen.com CANCER CELLBEHAVIOR • • • • • • Assays areprovidedfor: The CultreCoat CultreCoat CultreCoat Cancer CellAssays For theevaluationofaspecificmatrixcomponentCultreCoat Individual CultreCoat feature alsoallowsmultipleexperimentstobeconductedsimultaneouslyusingthesamekit. greater sensitivity, andaffordsflexibilityforthenumberofsamplesassessed.Thestripwell Fibronectin, andVitronectin.Theblackstripwellplateminimizesbackground,providing assess factorsthatinfluencecelladhesiononBME,Laminin,CollagenI,IV, CultreCoat non-specific binding. fluorescence assessedforeachwell.Controlsareprovideddeterminingbackgroundand the numberthatadhere,providingaloadingcontrolandpercent celladhesionbasedonthefinal Calcein -AMlabelingallowsdirectcomparisonsbetweenthenumberofcellsthatareloadedand background, providinggreatersensitivityandflexibilityforthenumberofsamplesassessed. for assessingfactorsthatinfluencecell-matrixinteractions.Theblackstripwellplateminimizes CultreCoat Cell attachmentisthencalculatedbydividingthefluorescencevalueforcellsloadedthatofattached. The unattachedcellsarethenwashedaway, andthecellsthathaveattachedisdeterminedbyfluorescenceoutput. precoated withECM.ThecellsattachtotheECMproteins,andtotalloadedisdeterminedbyfluorescenceoutput. Figure 1.TheTrevigen CellAdhesion Assay. Cellsarepre-labeledwithCalcein-AM,andloadedontostripwellplates Collagen I Vitronectin Fibronectin Collagen IV Laminin I Basement MembraneExtract(BME) ® ® ® Cell AdhesionAssaysareprovidedintwoformats: Cell AdhesionAssaysprovideasimple,standardized,96wellhighthroughputformat Cell AdhesionAssays ® ® Cell AdhesionProteinArray Cell AdhesionAssays ® Cell AdhesionAssays in one96-wellplateprovidesaformatto ® Cell Adhesion INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:18 PM Page 107

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Cancer Cell Assays CultreCoat® Cell Adhesion Assays CELL ADHESION ASSAYS OVERVIEW

Figure 5. Percent adhesion for MG63, human osteosarcoma, to Collagen IV, Laminin I, and BME, and HT1080, human fibrosarcoma, to Collagen I, Fibronectin, and Vitronectin. Samples were assessed in duplicate using 15,000 cells/well for an adhesion period of 1 hour and 15 minutes.

103 REFERENCES: 1. Dike, L.E. and D.E. Ingber, 1996. Integrin-dependent induction of early growth response genes in capillary endothelial cells. J Cell Sci 109:2855-63. 2. Zhu, X., et al., 1996. Adhesion-dependent cell cycle progression linked to the expression of cyclin D1, activation of cyclin E-cdk2, and phosphorylation of the retinoblastoma protein. J Cell Biol 133:391-403. 3. Takeuchi, Y., et al., 1997. Differentiation and transforming growth factor-beta receptor down-regulation by collagen-alpha2beta1 integrin interaction is mediated by focal adhesion kinase and its downstream signals in murine osteoblastic cells. J Biol Chem, 272:29309-16. 4. Xiao, G., et al., 2002. Bone morphogenetic proteins, extracellular matrix, and mitogen-activated protein kinase signaling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells. J Bone Miner Res, 17:101-10. 5. Cary, L.A., J.F. Chang, and J.L. Guan, 1996. Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J Cell Sci, 109:1787-94. 6. Hu, X.-W., D. Meng, and J. Fang, 2008. Apigenin Inhibited Migration and Invasion of Human Ovarian Cancer A2780 Cells through Focal Adhesion Kinase. Carcinogenesis, 29:2369-76. 7. Boudreau, N., et al., 1995. Suppression of ICE and apoptosis in mammary epi-thelial cells by extracellular matrix. Science, 267:891-3. 8. Haraguchi, M., et al., 2008. Snail regulates cell-matrix adhesion by regulation of the expression of integrins and basement membrane proteins. J. Biol. Chem, 283:23514-23.

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Cancer Cell Assays Cultrex® Cell Staining Kit

The Cell Staining Kit provides ready-to-use CS Solution (Catalog # 3437-100-01) and Wash Solution (Catalog # 3437-100-02). The CS Solution contains a mixture of Azure A and Methylene Blue specially formulated to provide optimized staining of cells and structures grown on BME. Ideal staining contrast is achieved with minimum background since excess stain can be easily washed away using the wash buffer. The blue coloration allows optimal contrast between cells and the growth matrix.

APPLICATIONS: Staining of cells grown on Cultrex® Basement Membrane Extract (BME) or other extracellular membrane protein extracts after applications such as: • Tube formation assay: Basement Membrane Extract promotes differentiation of endothelial cell lines (e.g. SVEC4-10), into capillary like structures. • Neurite outgrowth assay: Basement Membrane Extract promotes differentiation of neurites from chick dorsal root ganglion. • Aortic ring angiogenesis assay: Basement Membrane Extract promotes differentiation of rat or mouse aorta tissue to form branched, capillary-like structures that extend radially outward, and into the ring.

STORAGE:

SVEC4-10 cells stained with Cultrex® Cell Staining Kit. Cells Store at room temperature. were grown on Cultrex® BME, phenol red free in DMEM containing 6% FBS. QUALITY CONTROL: CELL STAINING KIT CELL STAINING • Each lot of cell staining solutions is inspected for, and must be free from, interfering particles. 104 • Each lot of cell staining solutions is functionally checked in staining of cells grown on Cultrex® Basement Membrane Extract. Cells must vividly contrast with the growth matrix and the field must be free from interfering background.

Cell Staining Kit, Human Umbilical Vein Endothelial Cells (HUVECs) stained with Cultrex® Cell Staining Kit. Cells were grown for four hours on gelled Cultrex® BME Reduced Growth Factor (catalog # 3433-005-02) in EGM-2 medium.

ORDERING INFORMATION

3437-100-K Cultrex® Cell Staining Kit, 100 ml each

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3-D Products 3-D Culture Matrices

Specialized cells that comprise tissues and organs are situated within a three-dimensional, dynamic and complex molecular framework known as the extracellular matrix, which is comprised of structural proteins and other components. These cells receive signals from receptor ligands within the extracellular matrix that influence key cell functions such as apical-basal polarization, invasion, migration, proliferation, and differentiation. Furthermore, normal cells that lose contact with the basement membrane undergo apoptosis, whereas malignant cells do not. Trevigen’s 3-D Culture Matrix™ products enable investigators to employ an innovative in vitro approach to modeling cell responses in in vivo-like environments, which cannot be observed in standard 2-D cultures.

To facilitate in vitro investigation of cell behavior in vivo we offer: 3-D CULTURE MATRICES • Cultrex® 3-D Culture MatrixTM BME • Cultrex® 3-D Culture MatrixTM Rat Collagen I • Cultrex® 3-D Culture MatrixTM Mouse Laminin I OVERVIEW To facilitate monitoring of cell proliferation in 3-D culture, we offer: • Cultrex® 3-D Culture 96 Well BME Cell Proliferation Assay Kit 3-D Culture of MCF-10A cells on 3-D Culture Matrix Collagen I in Assay • Cultrex® 3-D Culture 96 Well Collagen I Cell Proliferation Assay Kit Media* with 2% BME stained with SYBR® Green and Propidium Iodide. • Cultrex® 3-D Culture 96 Well Laminin I Cell Proliferation Assay Kit

*Assay Media is DMEM, 2% Horse Serum, 20mg/ml EGF, 500mg/ml Hydrocortisone, 100mg/ml Cholera Toxin, 10mg/ml Insulin, and 1X To retrieve cells cultured in 3-D BME or Laminin I for subsequent Pen/Strep. biochemical analysis we offer: • Cultrex® 3-D Culture MatrixTM Cell Harvesting Kit 10500 Cultrex® 3-D Culture Matrix™ Basement Membrane Extract (BME)

3-D Culture Matrix™ reduced growth factor (RGF) BME provides the foundation for cells to grow in three dimensions allowing for the formation of acinar and other hollow structures in vitro.

Specifications:

CONCENTRATION: 12–18 mg/ml.

SOURCE: Murine Engelbreth-Holm-Swarm (EHS) tumor.

STORAGE BUFFER: Dulbecco’s Modified Eagle’s medium containing 10 µg/ml gentamycin sulfate and no phenol red.

STORAGE/STABILITY: Non transgenic primary mammary cells grown in Cultrex® 3-D BME Product is stable for a minimum of 3 months from date of shipment when stored at -20°C in develop into a polarized acinus. Confocal microscopy (5 µm projection) a manual defrost freezer. For optimal stability, store at -80 °C in aliquots. demonstrates epithelial polarity: DAPI stain, blue: GM130, red (Golgi Avoid repeated freeze/thaw cycles. protein, apical marker; panel A), Z01, green (tight junctions, apical; panel B); Integrin a6, magenta (baso-lateral; panel C), overlay shown in panel D. Images courtesy of Martin Jechlinger. Material Qualification:

FUNCTIONAL ASSAY: • Acinar structure formation: 3-D Culture Matrix™ RGF BME promotes differentiation of a human ORDERING INFORMATION epithelial cell line derived from mammary gland (MCF-10A) into acinar structures. • Tube Formation Assay: BME promotes formation of capillary-like structures by human 3445-001-01 (HUVEC, HMVEC) or mouse (SVEC4-10) endothelial cells. Cultrex® 3-D Culture Matrix™ BME, 1 ml 3445-005-01 STERILITY TESTING: Cultrex® 3-D Culture Matrix™ BME, 5 ml • No bacterial or fungal growth detected after incubation at 37°C for 14 days following USP XXIV Chapter 71 sterility test. • No mycoplasma contamination detected by PCR. 1-800-873-8443 • www.trevigen.com • Endotoxin concentrations ≤ 8 EU/ml by LAL assay. INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:18 PM Page 110

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3-D Products 3-D Culture Matrix™ Mouse Laminin I

3-D Culture Matrix™ Mouse Laminin I may be used as a gel on which to grow cells, or as a media additive alone or in concert with other basement membrane components, to study cellular growth and differentiation in three dimensions in vitro. To offer the most standardized Laminin I for use in 3-D cultures, a special process is employed to provide material at a standard concentration of 6 mg/ml (by absorbance and extinction coefficient).

Specifications:

CONCENTRATION: 6 mg/ml MOUSE LAMININ I

TM PURITY: ≥90% (SDS-PAGE)

SOURCE: Murine Engelbreth-Holm-Swarm (EHS) tumor

STORAGE BUFFER: Dulbecco’s Modified Eagle’s medium containing 10 µg/ml gentamycin sulfate and no phenol red.

STORAGE/STABILITY:

3-D CULTURE MATRIX 3-D CULTURE Product is stable for a minimum of 3 months from date of shipment when stored at -20°C in a manual defrost freezer. For optimal stability, store at -80 °C in aliquots. 106 Avoid repeated freeze/thaw cycles. Material Qualification:

STERILITY TESTING: • No bacterial or fungal growth detected after incubation at 37°C for 14 days following USP XXIV Chapter 71 sterility test. • No mycoplasma contamination detected by PCR. • Endotoxin concentrations ≤ 20 EU/ml by LAL assay. Primary embryonic submandibular epithelium cultured in laminin-1 gel with FGF10 (200 ng/ml). The Epithelia are cultured for either 24 or 48 hours in serum-free media and undergo branching morphogenesis in culture. Image courtesy of Matthew P. Hoffman. Patel, V.N., Knox, S.M., Likar, K.M., Lathrop, C.A., Hossain, R., Eftekhari, S., Elkins, M., Vlodasky, I., Whitelock, J.M. and Hoffman, M.P. Heparanase cleavage of heparin sulfate modulates FGF10 function during submandibular gland branching morphogenesis. Development, 2007, 134(23):4177-86.

ORDERING INFORMATION

3446-005-01 Cultrex® 3-D Culture Matrix™ Mouse Laminin I, 30 mg

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3-D Products 3-D Culture Matrix™ Rat Collagen I

The 3-D Culture Matrix™ Collagen I may be used as a gel on which to grow cells or as a media additive alone or in concert with other basement membrane components, to study cellular growth and differentiation in three dimensions in vitro. Type I Collagen is the major structural 3-D CULTURE MATRIX™ RAT COLLAGEN I component of extracellular matrices found in connective tissue and internal organs, but is most prevalent in the dermis, tendons, and bone. It is a 300 kDa molecule composed of two alpha1(I) chains and one alpha2(I) chain that spontaneously forms a triple helix scaffold. This phenomenon can be exploited to promote cell attachment, growth, differentiation, migration, and tissue morphogenesis during development.

Specifications:

CONCENTRATION: 5 mg/ml (Sircol Assay)

SOURCE: OVERVIEW Rat tail tendons

STORAGE BUFFER: 20 mM Acetic Acid, pH 3.4 - 3.6

STORAGE/STABILITY: Product is stable for a minimum of 3 months from date of shipment if stored at 4°C. Do Not Freeze. 10700 Material Qualification:

ORDERING INFORMATION

3447-020-01 Cultrex® 3-D Matrix™ Rat Collagen I, 100 mg

1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 11/31/112:19PMPage112 108 3-D PROLIFERATION ASSAYS 1-800-873-8443 3447-096-K Cultrex 3447-096-K Cultrex 3446-096-K Cultrex 3445-096-K Cultrex 3445-096-CK Proliferation AssayKit Proliferation AssayKit Proliferation AssayKit Proliferation AssayCoreKit REIGINFORMATION ORDERING ® ® ® ® 3-D CultureCollagenICell 3-D CultureLamininICell 3-D CultureBMECell 3-D CultureBMECell • www.trevigen.com CANCER CELLBEHAVIOR Cultrex 3-D Products • tumorigenicity inan format forquantitatingthedegreetowhichpharmacologicalcompoundsinfluencetoxicityor tumorigenicity, andnewtumorformation.Theseassaysofferaflexible,standardized,high-throughput when usingcellmodelsinthescreeningprocessforcompoundsthatinfluencetoxicity, cellsurvival, Proliferation Assayswerecreatedinanefforttoprovidemorephysiologicallyrelevantassessments cell-based studiestoprovidethemostaccuratetranslationanimalmodels.Cultrex toxicity anddrugresistance.Asaresult,thisenvironmentmustbemimickedduringthecourseof aroleforextracellularproteinsininfluencingboth responses toapoptosisinducingagentsimplicating Recent studiesindicatethatthecompositionofextracellularenvironmentinfluencescellular The assayisavailableinthefollowingformats: to multipleformatssothatcellproliferationmaybeevaluatedagainstdifferentextracellularmatrices. Components arestoredatroomtemperatureand-20°C. STORAGE: cancer cells. 1. AoudjitF, Vuori K.(2001)Integrinsignalinginhibitspaclitaxel-inducedapoptosisinbreast REFERENCES: through b1integrin-dependentactivationofPI3-kinase. overrides DNAdamage-inducedcellcyclearrestandapoptosisinsmall-celllungcancercells 2. HodkinsonPS,ElliottT, Wong WS,RintoulRC,MackinnonAC,HaslettC,TSethi.(2006)ECM of untreatedcontrols. Reagent wasaddedtoeachwell,andabsorbanceat450nmdetermined2hours.Values wereassessedasapercentage treated aftertwohours.Cellcultureswereincubatedat37°C5%CO was conductedusingthetumorigenicityprotocol.Briefly, cellswereseededinthepresenceorabsenceofECMproteins and Proliferation ofMDA-MB-231cellsindifferentextracellularenvironmentsthepresence50 Basement MembraneExtract(BME) ® Oncogene 3-D ProliferationAssays in vivo in 20: -like environment.TheCultrex 4995-5004. , Laminin I,CollagenI. 2 Cell Death and Differentiation and Death Cell . for4days.Then15 ® Cell ProliferationAssayhasbeenadapted µ l of3DCultureCellProliferation µ l Etoposide.3DCulture 13: 1776–88. ® Cell INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:19 PM Page 113

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3-D Products Cultrex® 3-D Culture Cell Harvesting Kit

3-D Cultures exhibit cellular behaviors and morphologies similar to those seen in vivo; however, the adaptation of these models for studying biochemical processes has been impeded by the challenge of separating intact cells from extracellular proteins comprising the hydrogel. Commonly, proteases are employed to degrade these extracellular proteins; however, proteases also degrade proteins on the cell surface, and protease activity may carry over into lysate 3-D CULTURE CELL HARVESTING KIT preparations. Non-enzymatic methodologies have also been described for depolymerizing extracellular matrix proteins, although the implementation of these protocols remains problematic for some researchers. Trevigen’s Cultrex® 3-D Culture Cell Harvesting Kit provides an optimized and standardized solution for the isolation and normalization of cell lysates from 3-D Culture Matrix™ BME or Laminin I for subsequent biochemical analysis.

STORAGE: Components are stored at room temperature and -20°C. OVERVIEW

10900

Figure 1. Morphology of MCF-10A (A,B) and MCF-7 cells (C, D) in traditional 2-D culture and 3-D BME culture, scale = 250 µm.

ORDERING INFORMATION

3448-020-K Cultrex® 3-D Culture Cell Harvesting Kit, 20 Tests

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CANCER CELL BEHAVIOR

3-D Products Cultrex® 3-D Culture Cell Harvesting Kit 3-D CULTURE CELL HARVESTING KIT 3-D CULTURE

110

Figure 3. Evaluation of RNA expression via RT-PCR (A) and protein expression via Western blotting (B) for GAPDH from 1-800-873-8443 • www.trevigen.com MCF-10A and MCF-7 mammary epithelial cells isolated from either traditional 2D culture or 3D BME culture. INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:19 PM Page 115

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Cell Proliferation and Viability MTT Cell Proliferation Assay

The TACS® MTT Cell Proliferation Assay (MTT-CPA) is a sensitive kit for the measurement of cell proliferation based upon the reduction of the tetrazolium salt, 3,[4,5-dimethylthiazol-2- yl]-2,5-diphenyl-tetrazolium bromide (MTT). Changes in cell proliferative activity caused by trophic factors, growth inhibitors, or inducers and inhibitors of apoptosis, may be quantified using the MTT-CPA. MTT is reduced to an insoluble formazan dye by mitochondrial enzymes CELL PROLIFERATION AND VIABILITY associated with metabolic activity. The reduction of MTT is primarily due to glycolytic activity within the cell and is dependent upon the presence of NADH and NADPH. (See p.145)

STORAGE: Components are stored at 4°C and room temperature. XTT Cell Proliferation Assay

The XTT Cell Proliferation Assay (XTT-CPA) is a sensitive kit for the measurement of cell OVERVIEW proliferation based upon the reduction of the tetrazolium salt, 2,3-Bis(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide. Cleavage of the tetrazolium salt to formazan occurs via the succinate-tetrazolium reductase system in the mitochondria of metabolically active cells. The reaction is attributed mainly to mitochondrial enzymes and electron carriers, but a number of other non-mitochondrial enzymes have been implicated. (See p.146)

STORAGE: Components are stored at -20°C. 11100 Calcein-AM Cell Viability Assay

3-D Culture of MCF-10A mammary endothelial cells on The Calcein-AM Kit provides a simple, rapid and accurate method to measure cell viability 3-D Culture Matrix™ BME in Assay Medium* with 2% BME and/or cytotoxicity. Calcein-am is a non-fluorescent, hydrophobic compound that easily and stained with Calcein-AM. permeates intact, live cells. The hydrolysis of Calcein-AM by intracellular esterases produces calcein, a hydrophilic, strongly fluorescent compound that is well-retained in the cell cytoplasm. Cells grown, preferably in black-walled plates, can be stained and quantified in less than two hours. (See page 147)

STORAGE: Components are stored at -20°C and 4°C.

*Assay Medium: DMEM, 2% Horse Serum, 20 ng/ml EGF, 500 ng/ml Hydrocortisone, 100 ng/ml Cholera Toxin, 10 µg/ml Insulin, and 1X Pen/Strep.

REFERENCES: 1. Genoveva Murillo, Xinjian Peng, Karen E.O. Torres, and Rajendra G. Mehta. (2009) Deguelin Inhibits Growth of Breast Cancer Cells by Modulating the Expression of Key Members of the ORDERING INFORMATION Wnt Signaling Pathway. Cancer Prevention Research 2: 942 - 950. 2. Kyung-Hee Chun, Jerome W. Kosmeder, II, Shihua Sun, John M. Pezzuto, Reuben Lotan, 4890-025-K MTT Cell Proliferation Assay Kit, Waun Ki Hong, and Ho-Young Lee. (2003) Effects of Deguelin on the Phosphatidylinositol 2500 Wells 3-Kinase/Akt Pathway and Apoptosis in Premalignant Human Bronchial Epithelial Cells.J Natl Cancer Inst 95: 291 - 302. 4890-050-K 3. Dalit Barkan, Lara H. El Touny, Aleksandra M. Michalowski, Jane Ann Smith, Isabel Chu, MTT Cell Proliferation Assay Kit, Anne Sally Davis, Joshua D. Webster, Shelley Hoover, R. Mark Simpson, Jack Gauldie, and 5000 Wells Jeffrey E. Green. (2010) Metastatic Growth from Dormant Cells Induced by a Col-I–Enriched 4891-025-K Fibrotic Environment. Cancer Res 70: 5706 - 5716. XTT Cell Proliferation Assay Kit, 2500 Wells 4892-010-K Calcein-AM Cell Viability Assay Kit, 1000 Wells

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DNA Damage and Genomic Instability APOPTOSIS DETECTION

Cancer Cell Behavior

APOPTOSIS DETECTION

Stem Cell Products

Oxidative Stress

Molecular Biology Reagents APOPTOSIS DETECTION APOPTOSIS INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:19 PM Page 118

APOPTOSIS DETECTION

Overview

Apoptosis was originally defined by the morphological changes that occur with remarkable fidelity in a wide variety of cell types, regardless of cell lineage and the method of cell death induction. The morphological changes can occur rapidly after induction and include cell surface blebbing, cell and nuclear shrinkage. The morphological changes are best described in Apoptotic thymocytes in vitro, and Figure 1 provides an illustration of typical cell morphologies that may Pathway be observed during a 24 hour period after induction of apoptosis. Consideration of morphology Necrotic Pathway is the single most important method for detection of apoptosis and no other biochemical test or assay is available to replace microscopic observation.

Researchers require alternative methods for scoring apoptosis that provide supportive biochemical evidence or quantitation of data. This can be particularly important in vivo when the later, more obvious morphological changes may not occur due to phagocytosis and cell removal, and in experimental systems when large sample numbers have to be assayed and screened. Once morphological evidence has determined that the mechanism of cell death occurs by apoptosis, continued stringent analysis using morphology is not necessary and a more convenient biochemical method is more appropriate. As elucidation of the complex and varied biochemical pathways leading to the execution of apoptosis continues, it is likely that the options available for apoptosis detection will continue to expand. Figure 1: A schematic representation of cells undergoing apoptosis (clockwise from top) or necrosis (counterclockwise Please check our website for the latest product additions and product information. APOPTOSIS DETECTION OVERVIEW from top). Apoptotic cells undergo a series of morphological changes including chromatin condensation and fragmentation, 114 membrane blebbing, and ultimately break-up and engulfment by surrounding vital cells. Necrotic cells are characterized by a breakdown of nuclear, cytoplasmic and lysosomal membranes resulting in the swelling and breakage of the cell. Cell Membrane Events

PRODUCTS OFFERED: Annexin V Conjugates

Some of the earliest apoptotic changes occur at the cell surface. The early recognition of apoptotic cells by phagocytic cells, the significant loss of water leading to cell shrinkage, and the maintenance of intact cell membranes, despite the cell surface blebbing observed in many cell types, indicate significant changes have occurred in the plasma membrane. One of the better understood cell surface modifications is exposure of phosphatidylserine (PS). Normally, PS is confined to the inner layer of the lipid bilayer. This asymmetric distribution is maintained in normal cells by the action of specific proteins. After induction of apoptosis under the influence of translocases, the PS is flipped from the inner to the outer bilayer, rendering the molecule available for detection. Annexin V is a blood clotting factor that exhibits a high calcium-dependent specificity for PS binding. The coupling of annexin to fluorescein, or biotin, generates a direct, rapid, and simple method for the detection of apoptosis on unfixed cells. Figure 2 is a depiction of PS flipping during apoptosis and subsequent detection using Annexin V.

Figure 2: Schematic depiction of phosphatidylserine flipping during apoptosis and subsequent detection using

Ca++ annexin V. Ca++ ++ Cell Ca ++ Ca Membrane

Outer Apoptosis

Inner

Phosphatidylserine Other Phospholipids Annexin V

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APOPTOSIS DETECTION

Mitochondrial-Related Events

PRODUCTS OFFERED: anti-cleaved caspase-3, anti-PARP, PARP Apoptosis Assay Kits,Bcl-2 family antibodies, DePsipher™, MitoShift™, PBR Protein, anti-PBR

Although organelles remain grossly normal during the earlier stages of apoptosis, research has revealed a pivotal role for mitochondria in the apoptotic cascade. The energy generated during cellular respiration accumulates in the mitochondrial transmembrane space as an electron gradient APOPTOSIS DETECTION OVERVIEW called the mitochondrial membrane potential or Δψm. This electrochemical gradient is often disturbed during apoptosis and can be detected using cationic dyes such as DePsipher™ (5,5’6,6’- tetrachloro-1,1’,3,3’-tetraethylbenz-imidazolylcarbocyanine iodide) or MitoShift™ (tetramethylrhodamine ethyl ester). DePsipher™ readily enters cells and accumulates as a multimeric aggregate within healthy mitochondria, and under UV light, this multimeric form emits red light. In apoptotic Figure 3: Identification of apoptotic cells using DePsipher™. cells, the mitochondrial membrane potential collapses and the dye returns to a monomeric form. INT407 human cells were treated with 25 µM etoposide for 6 hours, and then incubated with the DePsipher™ reagent in The monomeric molecule emits green fluorescence under UV light. DePsipher™ provides a rapid Reaction Buffer for 30 minutes prior to visualization. Healthy fluorescence-based assay for the apoptosis-associated loss of mitochondrial potential (Figure 3). OVERVIEW cells (containing red aggregates) can be differentiated from apoptotic cells (containing mostly green monomers) Activation of caspases, or cysteinyl proteases, is a necessary event for execution of the apoptotic response. Some of the caspases are activated early in the apoptotic process and their activation is the first step in a cascade of proteolytic cleavage of key proteins and enzymes, including other caspases and poly (ADP-ribose) polymerase (PARP). The caspases each cleave a defined amino acid sequence. This specificity has led to the development of highly specific irreversible peptide inhibitors. Delivery of these peptides allows for complete inhibition in the execution of apoptosis, thereby providing a means to probe the early events in apoptosis and to investigate the ordering of key events in the process. Since the substrate specificity of the caspases is high, analysis of substrate cleavage also provides a useful biochemical marker. Figure 4 shows a Western blot 11500 detecting cleaved PARP in apoptotic cells compared to a population of mostly healthy cells. Figure 5 shows the presence of cleaved caspase-3 in Jurkat cells after treatment to induce apoptosis. (see page 116).

The Bcl-2 family of proteins is well known to be important in determining cell fate, although their mechanism of action remains unclear. The relative levels of the family members, as well as the subcellular distribution of these proteins, changes during apoptosis. In particular, the movement of some members from the cytoplasm to the mitochondria and the subsequent associations that occur between the Bcl-2 family members and other mitochondrial membrane associated proteins are indicated to be crucial steps in apoptosis. Antibodies that recognize the individual Bcl-2 family members provide powerful tools for studying alterations in expression levels, the subcellular redistribution, and protein associations of these intriguing molecules.

Figure 4: Western blot analysis of Wehi, Jurkat and CCRF-CEM cell lines untreated (H) and treated (T) with 25 µM Etopo-side for 16 hours at 37oC. Cells were lysed in Tris-Glycine SDS sample buffer at the concentration 1 x 107 cells/ml and 10 µl of each lysate were loaded per well of a 4-20% Tris-Glycine gel. Proteins were transferred onto an Immobilon FL membrane and PARP was detected with Trevigen’s anti-PARP (C2-10) antibody followed by an IR800-conjugated secondary antibody. The membrane was scanned using an Odyssey Infrared Imaging System (Licor). The 116 kDa band corresponds to full length PARP and the 85 kDa band is the apoptotic cleavage product.

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APOPTOSIS DETECTION

DNA Fragmentation

PRODUCTS OFFERED: Anti-γH2AX antibody, apoptotic DNA laddering kit, TACS•XL®, TACS® 2, VasoTACS™, FlowTACS™, TiterTACS™ 96 Well Kit, NeuroTACS™ II, TumorTACS™, CardioTACS™, DermaTACS™

DNA fragmentation occurs as one of the final stages of cell death and has long been considered a hallmark of apoptosis and one of the defining biochemical events of the pathway. DNA fragments are generated initially through single-stranded breaks that produce fragments of DNA larger than 50,000 bases. Later in the process, double-stranded DNA cleavage occurs mainly in the linker regions between nucleosomes. The length of DNA wrapped around the histone core within nucleosomes is approximately 200 bases. The cleavage generates DNA ends with a free 3’ hydroxyl group. For detection of double-strand DNA breaks, anti-γH2AX is unique in only detecting phosphorylated histones at sites of double-strand DNA breaks. Figure 5: Jurkat cells were treated with 25 µM etoposide γ for 5 hours to induce apoptosis. Cells were labeled with Trevigen’s phosphorylated H2AX antibody has been qualified for both Western blotting and 1:250 dilution of anti-cleaved caspase-3 followed by immunohistochemistry. anti-rabbit horseradish peroxidase. Detection performed with Trevigen’s TACS® Blue Label™ and counterstained For detection of the DNA fragmentation associated with apoptosis by DNA laddering, the DNA with Nuclear Fast Red. is isolated and the cleaved fragments are separated by agarose gel electrophoresis. If sufficient DNA is present, staining of the gel with ethidium bromide reveals the typical laddering pattern of multimers of 180–220 bases. Trevigen’s Ethidium Bromide DNA Laddering kit provides the necessary reagents for detection of the DNA ladder.

APOPTOSIS DETECTION OVERVIEW DNA fragmentation can also be detected within the nuclei of cells and tissues. Many of the standard fixatives used for preserving cells and tissues maintain the integrity of the DNA and 116 the free 3’ hydroxyl groups at the sites of cleavage. The DNA thus provides a target for terminal deoxynucleotidyl transferase (TdT), which can sequentially add nucleotides to the 3’ end of DNA. The addition of a labeled nucleotide (i.e. biotin dUTP or BrdU) in the reaction mix then allows indirect visualization of any DNA labeling by TdT within fixed cells or tissue sections. Utilizing a TUNEL-based assay, Trevigen has developed a series of kits for the in situ detection of apoptosis with colorimetric and fluorometric options. The TACS® kits are tailored for the detection of DNA fragmentation associated with apoptosis in a variety of cell and tissue types and for analysis by different formats that include microscopy, flow cytometry, and 96 well plates.

*TACS®: Trevigen Apoptotic Cell System*

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APOPTOSIS DETECTION

Assay Selection Guide APOPTOSIS ASSAY SELECTION GUIDE C a t a l o g # D e s c r i p t i o n Light MicroscopyFluorescent Flow Microscopy CytometryGel Electrophoresis Plate ReaderDouble LabelConfocal UnfixedMicroscopy CellsFixed CellsParaffin EmbeddedFrozen TissueFresh Tissue TissuePage 4828-30-DK TACS•XL® DAB1 121 4828-30-BK TACS•XL® Blue Label 121 4810-30-K TACS® 2 TdT DAB 123 4811-30-K TACS® 2 TdT Blue Label 123 4812-30-K TACS® 2 TdT Fluorescein 123 4827-30-K CardioTACS™ 126 4829-30-K DermaTACS™ 128 4823-30-K NeuroTACS™ II 130 4822-96-K TiterTACS™ 135 4815-30-K TumorTACS™ 132 4826-30-K VasoTACS™ 134 4817-60-K FlowTACS™ 137 4830-01-K (100 tests) TACS® Annexin V-FITC 118 4830-250-K (250 tests) 4835-01-K (100 tests) TACS® Annexin V-Biotin2 3 120 4835-250-K (250 tests) 6300-100-K DePsipher™ 140 6305-100-K MitoShift ™ 142 117 4253-096-K CometAssay® 25 4850-20-ET TACS® DNA Laddering Kit 139 4859-20-K TACS® DNA Tissue 139 Supplement Kit 4684-096-K (HT Colorimetric) PARP/Apoptosis Assay 144 4685-096-K (HT Chemiluminescent) 4891-025-K MTT Cell Proliferation Assay 145 4891-025-K XTT Cell Proliferation Assay 146 4892-010-K Calcein AM Cell Viability Kit 147

1TACS®: Trevigen Apoptotic Cell System.

2Annexin V-Biotin can be fixed AFTER binding to live cells.

3Please see instructions for use.

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APOPTOSIS DETECTION

Annexin V Assays TACS® Annexin V-FITC Kit

The TACS® Annexin V-FITC Kit allows rapid, specific, and quantitative identification of apoptosis in individual cells. Annexin V is a calcium-dependent phospholipid binding protein. During apoptosis, an early and ubiquitous event is the exposure of phosphatidylserine at the cell surface. The annexin V is supplied with an optimized Binding Buffer and propidium iodide. Propidium iodide may be used on unfixed samples to determine the population of cells that have lost membrane integrity, an indication of late apoptosis or necrosis.

FEATURES: • Fast, sensitive detection of apoptotic cells. • Includes propidium iodide for discriminating apoptotic and necrotic (or late apoptotic) cells.

APPLICATIONS: • Flow cytometry. • In situ detection of apoptotic cells.

COMPONENTS: 4830-01-K TACS® Annexin V-FITC Kit.

Catalog # Component Size 4830-01-1 TACS® Annexin V-FITC* 100 µl 4830-01-2 10X Binding Buffer 8 ml ANNEXIN V-FITC 4830-01-3 Propidium Iodide** 1 ml

118 4830-250-K TACS® Annexin V-FITC Kit

Catalog # Component Size 4830-250-1 TACS® Annexin V-FITC* 250 µl 4830-250-2 10X Binding Buffer 20 ml 4830-250-3 Propidium Iodide** 2.5 ml

* Excitation/Emission (492/520 nm) ** Excitation/Emission (480–530/>600 nm)

RELATED PRODUCTS: • TACS® Annexin V-Biotin Kit • DePsipher™ Mitochondrial Potential Assay • MitoShift™ Mitochondrial Potential Assay • FlowTACS™ Kit • TACS® 2 TdT FITC Kit

STORAGE: Store at 4˚C.

REFERENCES: 1. Kaetzel, M.A. and J.R. Dedman. 1995. Annexins: novel Ca2+ dependent regulators of membrane function. NIPS 10:171-174. 2. Koopman, G., C.P.M. Reutelinsperger, G.A.M. Kuijten, R.M.J. Keehnen, S.T. Pals, and M.H.J. van Oers. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84(5):1415-1420. 3. Martin, S.J., C.P.M. Reutelinsperger, A.J. McGahon, J.A. Rader, R.C.A.A. van Schie, D.M. LaFace, ORDERING INFORMATION and D.R. Green. 1995. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and 4830-01-K Abl. J. Exp. Med. 182:1545-1556. Annexin V-FITC Kit, 100 Tests 4830-250-K Annexin V-FITC Kit, 250 Tests

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APOPTOSIS DETECTION

Annexin V Assays TACS® Annexin V-FITC Kit

RECENT CITATIONS: Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation Rodrigo Franco, Mihalis I. Panayiotidis, and John A. Cidlowski J. Biol. Chem., Oct 2007; 282(42): 30452-65.

Selective Regulation of Bone Cell Apoptosis by Translational Isoforms of the Glucocorticoid Receptor Nick Z. Lu, Jennifer B. Collins, Sherry F. Grissom, and John A. Cidlowski Mol. Cell. Biol., Oct 2007; 27(20): 7143-60.

MAPK and heat shock protein 27 activation are associated with respiratory syncytial virus induction of human bronchial epithelial monolayer disruption

Divyendu Singh, Kelly L. McCann, and Farhad Imani ANNEXIN V-FITC Am J Physiol Lung Cell Mol Physiol, Aug 2007; 293: L436 - L445.

Effects on neurite outgrowth and cell survival of a secreted fibroblast growth factor binding protein upregulated during spinal cord injury Elena Tassi, Sharon Walter, Achim Aigner, Rafael H. Cabal-Manzano, Ranjan Ray, Paul J. Reier, and Anton Wellstein

Am J Physiol Regulatory Integrative Comp Physiol, Aug 2007; 293: R775 - R783. 119

Analysis of dexamethasone untreated thymocytes (A) and thymocytes treated with 100 nM dexamethasone for 15.5 hrs (B) using TACS® Annexin V-FITC and propidium iodide. The results show a distinct population of cells that have bound Annexin V (lower right quadrant of dot plot). These cells are early apoptotic. Annexin V positive cells that also take up propidium iodide are either late B apoptotic or necrotic (upper right quadrant of dot plot). There is also a population negative for both Annexin V and Propidium iodide (lower left quadrant of dot plot). These are normal viable cells. Analysis courtesy Dr. C. M. Knudson, Howard Hughes Medical Institute, St. Louis, MO.

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APOPTOSIS DETECTION

Annexin V Assays TACS® Annexin V-Biotin Kit

The TACS® Annexin V-Biotin Kit allows rapid, specific, and quantitative identification of apoptosis in individual cells. Annexin V is a calcium-dependent phospholipid binding protein. During apoptosis, an early and ubiquitous event is the exposure of phosphatidylserine at the cell surface. The annexin V is supplied with an optimized Binding Buffer and propidium iodide. Propidium iodide may be used on unfixed samples to determine the population of cells that have lost membrane integrity, an

Counts indication of late apoptosis or necrosis. Both early and late apoptotic cells in tissues, which are either cultured or freshly isolated, can be detected using TACS® Annexin V-Biotin.

FEATURES: • Fast, sensitive detection of apoptotic cells.

0 70 140 210 280 350 • Includes propidium iodide for discriminating apoptotic and necrotic (or late apoptotic) cells. • Provides flexibility in fluorophore choice (see page 158).

Flow cytometry analysis of WEHI 7.1 cells labeled with Annexin APPLICATIONS: V-Biotin and detected by streptavidin-FITC. WEHI 7.1 cells treated • Flow cytometry with 25 µM etoposide for two hours, with an overnight recovery, • In situ detection of apoptotic cells produce a peak approximately at log of 103 in the fluorescence channel 1 (FL1) (samples 2 and 3). Healthy WEHI 7.1 cells produce • Can be fixed after binding to live cells a peak at less than log 101 in the FL1 channel, which is similar to unlabeled cells (samples 4,5, and 1 respectively). Two different COMPONENTS: populations of cells (samples 1,2,4 and 3,5 respectively), 4835-01-K TACS® Annexin V-Biotin Kit were analyzed.

ANNEXIN V-BIOTIN Catalog # Component Size 4835-01-1 TACS® Annexin V-Biotin 100 µl 120 4830-01-2 10X Binding Buffer 8 ml 4830-01-3 Propidium Iodide 1 ml

4835-250-K TACS® Annexin V-Biotin Kit

Catalog # Component Size 4835-250-1 TACS® Annexin V-Biotin 250 µl 4830-250-2 10X Binding Buffer 20 ml 4830-250-3 Propidium Iodide 2.5 ml

RELATED PRODUCTS: • TACS® Annexin V-FITC Kit • DePsipher™ Mitochondrial Potential Assay • MitoShift™ Mitochondrial Potential Assay • FlowTACS™ Kit • TACS® 2 TdT FITC Kit

STORAGE: Store at 4˚C.

REFERENCES: 1. Kaetzel, M.A. and J.R. Dedman. 1995. Annexins: novel Ca2+ dependent regulators of membrane function. NIPS 10:171-174. 2. Koopman, G., C.P.M. Reutelinsperger, G.A.M. Kuijten, R.M.J. Keehnen, S.T. Pals, and M.H.J. van Oers. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84(5):1415-1420. 3. Martin, S.J., C.P.M. Reutelinsperger, A.J. McGahon, J.A. Rader, R.C.A.A. van Schie, D.M. LaFace, ORDERING INFORMATION and D.R. Green. 1995. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and 4835-01-K Abl. J. Exp. Med. 182:1545-1556. Annexin V-Biotin Kit, 100 Tests 4. J Baleriola, J., T. Suárez, and E.J. de la Rosa. 2010. DNA-PK promotes the survival of young neurons in the embryonic mouse retina. Cell Death Differ. 2010 May 7. [Epub ahead of print]. 4835-250-K Annexin V-Biotin Kit, 250 Tests

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APOPTOSIS DETECTION

DNA Fragmentation TACS•XL® In situ Apoptosis Detection Kits

TACS•XL® embodies a more robust approach for the in situ detection of apoptosis than other methods. The TACS•XL® Kit is based on incorporation of bromodeoxyuridine (BrdU) at the 3’ OH ends of the DNA fragments that are formed during apoptosis. The incorporation of BrdU by TdT is more efficient than either biotinylated or digoxigenin labeled nucleotides used in other terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-based assays. The detection system utilizes a biotin conjugated anti-BrdU antibody and streptavidin-horseradish peroxidase. The combination of antibody specificity with the signal enhancing properties of biotin streptavidin results in precise cellular labeling and the highest signal-to-noise ratio Detection of apoptotic cells in the photoreceptor layer of observed in competitive testing. retina in 10-day-old FVB mice using the TACS•XL® DAB kit. The tissue was fixed in 10% formalin and 5 µM paraffin Three kit formats are available: TACS•XL® DAB, TACS•XL® Blue Label, and TACS•XL® Basic. sections were used for the assay (400X magnification). The TACS•XL® DAB Kit is supplied with DAB and Methyl Green counterstain, and the TACS•XL® Photo courtesy Dr. S. Alikunju, Department of Cell Biology, Baylor College of Medicine, Houston, TX. Blue Label Kit is provided with TACS Blue Label™ and Nuclear Fast Red counterstain. These complete kits provide all the reagents required for labeling including two permeabilization reagents, labeling and detection reagents, stop buffers, counterstain and TACS-Nuclease™ reagents for generating positive controls with your own samples. The TACS•XL® Basic Kit provides the reagents necessary for routine labeling, and is ideal for researchers whose labs are equipped for standard immunohistochemical procedures involving horseradish-peroxidase. TACS•XL Please refer to our components listing for details.

FEATURES: ® • High signal-to-noise ratio generates stronger signal with less background. • Less sensitive to protease-induced false positive labeling than digoxigenin or biotin-based kits. • Complete kit provides either DAB or TACS Blue Label™ detection options. 121 • Includes exclusive Cytonin™ permeabilization reagent. • Includes TACS-Nuclease™ control reagents. • Readily adapted for fluorescence read-out.

APPLICATIONS: • In situ detection of apoptosis (by TUNEL) in fixed frozen or paraffin embedded tissue sections. • Assists in the identification of apoptotic morphologies.

ITEMS NOT INCLUDED:

Reagents for dehydration and rehydration of paraffin embedded sections, H2O2, methanol, PBS buffer, mounting medium, and disposables.

STORAGE: ORDERING INFORMATION Store components at -20˚C, 4˚C, and room temperature. 4828-30-K TACS•XL® Basic Kit, 30 Samples 4828-30-BK TACS•XL® Blue Label Kit, 30 Samples 4828-30-DK TACS•XL® DAB Kit, 30 Samples 4828-30-R TACS•XL® Replenisher Kit, 30 Samples 4828-30-AC TACS•XL® Antibody Module, 30 samples 4828-30-BC Blue Label Detection Module 4828-30-DC DAB Detection Module 4828-30-N Nuclease Module

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APOPTOSIS DETECTION

DNA Fragmentation TACS•XL® In situ Apoptosis Detection Kits

REFERENCES: 1. Murray, N., L.A. Davidson, R.S. Chapkin, W.C. Gustafson, D.G. Schattenberg, and A.P. Fields. 1999. Overexpression of protein kinase C II induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis. J. Cell. Biol. 45:699-711. 2. Bank, N., M. Kiroycheva, P.C. Singhal, G.M. Anthony, G.J. Southan, and C. Szabo. 2000. Inhibition of nitric oxide synthase ameliorates cellular injury in sickle cell mouse kidneys. Kidney Int. 58:82-89. 3. Li, X. and Z. Darzynkiewicz. 1995. Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Prolif. 28:571-579. 4. Gavrieli. Y., Y. Sherman, and S.A. Ben-Sasson. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell. Biol. 119:493-501.

PRODUCT CITATION: 1. Wang, K.X., Y. Shi, and D.T. Denhardt. 2007. Osteopontin regulates hind limb unloading- induced lymphoid organ atrophy and weight loss by modulating corticosteroid production Proc. Natl. Acad. Sci. USA 104:14777-14782, ® COMPONENTS: TACS•XL

122

Catalog # Component Size 4828-30-K - 4828-30-BKTACS•XL Basic 4828-30-DK- TACS•XL Blue 4828-30-R- TACS•XL Label -DAB 4828-30-BCTACS•XL Replenisher 4828-30-DC- Blue Label4828-30-N DABDetection - Detection Module- Nuclease Module Module 4800-30-01 Proteinase K 30 µl • • • • 4876-05-01 Cytonin™ 5 ml • • • • 4800-30-15 TACS-Nuclease™ 15 µl • • • 4800-30-16 TACS-Nuclease Buffer 1.5 ml • • • 4810-30-02 10X TdT Labeling Buffer 100 ml • • • 4810-30-03 10X TdT Stop Buffer 100 ml • • • 4810-30-05 TdT Enzyme 30 µl • • • • 4828-30-04 BrdU-dNTP Mix 30 µl • • • • 4828-30-06 Anti-BrdU Antibody 30 µl • • • • 4800-30-06 Streptavidin-HRP 30 µl •••• 4828-30-12 Streptavidin-Diluent 7.5 ml • • • • 4800-30-11 TACS Blue Label™ 3 ml • • 4800-30-17 Nuclear Fast Red 50 ml • • 4800-30-07 DAB Solution 3.75 ml • • 4800-30-09 DAB Enhancer 1 ml • • 4828-30-18 1% Methyl Green Solution 50 ml • •

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APOPTOSIS DETECTION

DNA Fragmentation TACS® 2 TdT In situ Apoptosis Detection Kits

TACS® 2 TdT Kits utilize Trevigen’s unique cation optimization system to enhance labeling within particular tissues. The TACS® 2 TdT Kits also employ a proprietary labeling buffer that contains no toxic components (e.g. sodium cacodylate). A highly purified form of the TdT enzyme is included for the enzymatic incorporation of biotinylated nucleotides. Biotin labeling is detected using streptavidin-horseradish peroxidase, and colorimetric substrates diaminobenzidine (DAB), or TACS Blue Label™. For fluorescent detection, a fluorescein conjugate of streptavidin is used and visualized by epifluorescence microscopy.

These complete kits provide all the reagents required for labeling including two permeabilization reagents, labeling and detection reagents, stop buffers, counterstain, and TACS-Nuclease™ Detection of apoptosis in post-weaning mouse breast tissue reagents for generating positive controls with your own samples. Please refer to our components using the TACS® 2 TdT DAB kit. Tissue sections were collected and fixed in 10% neutral buffered formalin, listing for details. embedded in paraffin and sectioned at 5 µM. Deparaffinized and rehydrated sections were processed following the The core kit is for researchers familiar with in situ labeling who are already set up for TACS® 2 TdT protocol. immunohistochemistry. A replenisher kit is also available for users of our TACS® kits who have used up the limiting reagents within their kit. The core kit provides a cost effective

replacement of the reagents that are used most quickly. TACS

FEATURES: ®

• TdT based in situ labeling. 2 TdT • Cation optimization for high signal and low background. • Complete kit provides either DAB, TACS Blue Label™, or fluorescence detection options. • Includes exclusive Cytonin™ permeabilization reagent. • Includes TACS-Nuclease™ control reagents. 123 APPLICATIONS: • In situ detection of apoptosis (by TUNEL) in fixed frozen, paraffin embedded, or plastic embedded cells and tissues. • Light microscopy • Fluorescence microscopy • Flow cytometry

ITEMS NOT INCLUDED:

Reagents for dehydration and rehydration of paraffin embedded sections, H2O2, methanol, PBS buffer, mounting medium, and disposables.

STORAGE: Components are stored at -20˚C, 4˚C, and room temperature.

ORDERING INFORMATION

4810-30-CK TACS® 2 TdT Core Kit, 30 Samples 4810-30-K TACS® 2 TdT DAB Kit, 30 Samples 4810-30-R TACS® 2 TdT Replenisher Kit, 30 Samples 4811-30-K TACS® 2 TdT Blue Label Kit, 30 Samples 4812-30-K TACS® 2 TdT Fluorescein Kit, 30 Samples

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APOPTOSIS DETECTION

DNA Fragmentation TACS® 2 TdT In situ Apoptosis Detection Kits

REFERENCES: 1. Cooper, L.F., J.C. Tiffee, J.P. Griffin, H. Hamano, and Z. Guo. 2000. Estrogen-induced resistance to osteoblast apoptosis is associated with increased hsp27 expression. J. Cell Physiology 185:401-407. 2. Kasahara, Y., R. Tuder, L. Taraseviciene-Stewart, T. LeCras, S. Abman, P. Hirth, J. Waltenberger, and N. Voelkel. 2000. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J. Clin. Inves. 106:1311-1319. 3. Gavrieli, Y., Y. Sherman, and S.A. Ben-Sasson. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell. Biol. 119:493-501. 4. Lovelace, C.I.P., J. Zhang, P.G. Vanek, and G.B. Collier. 1996. Detecting apoptotic cells in situ. Biomedical Products 21:76-77.

PRODUCT CITATION: 1. Tao, Y., I. Zafar, J. Kim, R.W Schrier, and C.L. Edelstein. 2008. Caspase-3 gene deletion prolongs survival in polycystic kidney disease. J. Am. Soc. Nephrol. 19:749-755.

COMPONENTS: 2 TdT ® TACS

124

Catalog # Component Size 4810-30-K TACS4811-30-K 2 TdT TACSDAB4812-30-K 2 Blue TACS 4810-30-CKLabel 2 Fluorescein4810-30-R TACS 2 Core TACS Kit 2 Replenisher 4800-30-01 Proteinase K 30 µl • • • • 4876-05-01 Cytonin™ 5 ml • • • 4800-30-15 TACS-Nuclease™ 15 µl • • • 4800-30-16 TACS-Nuclease Buffer 1.5 ml • • • 4810-30-02 10X TdT Labeling Buffer 100 ml • • • • 4810-30-03 10X TdT Stop Buffer 100 ml • • • • 4810-30-04 TdT dNTP Mix 30 µl • • • • • 4810-30-05 TdT Enzyme 30 µl • • • • • 4800-30-06 Streptavidin-HRP 30 µl • • •• 4800-30-14 Streptavidin-Fluorescein 30 µl • 4800-30-07 DAB Solution 3.75 ml • 4800-30-09 DAB Enhancer 1 ml • 4800-30-11 TACS Blue Label™ 3 ml • 4800-30-12 Blue Streptavidin-Diluent 7.5 ml • 4800-30-18 1% Methyl Green Solution 50 ml • 4800-30-17 Nuclear Fast Red 50 ml • 4810-30-09 50X Co+2 30 µl • • • • • 4810-30-10 50X Mg+2 30 µl • • • • • 4810-30-14 50X Mn+2 30 µl • • • • •

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APOPTOSIS DETECTION

DNA Fragmentation Specialty In Situ Apoptosis Detection

Through our own extensive in-house testing and the information we obtained from cells and tissues processed through our labeling services, Trevigen has developed specialty kits that are qualified and optimized for use with specific tissues. We removed the “guesswork” and the time-consuming optimization that may be required for the first time user. We provide tailor-made kits for apoptosis detection in specific cell and tissue types. These include neuronal cells and tissues, tumor tissues, cardiac-derived samples, vascular-derived samples and skin. These kits provide the appropriate permeabilization reagent for the tissue under investigation with recommendations on incubation times and temperatures to ensure that the labeling enzyme has optimal access to nuclear DNA. Each kit provides a preselected cation for optimal labeling of free apoptotic DNA ends, and minimized labeling of necrotic cells. The method for visualization of the labeled cells provides a low background and ease of interpre- SPECIALTY tation in the tissue selected, and the counterstaining reagents have been prequalified to ensure nominal overlap between labeled and unlabeled cells, and for interpretation of morphology in the fully processed sample.

For the researcher that is studying apoptosis in specific tissue types, we have the CardioTACS™, IN SITU NeuroTACS™ II, TumorTACS™, DermaTACS™, and VasoTACS™ kits. Each has been rigorously qualified and field tested for tissue-specific labeling and incorporates all the benefits discussed above. Trevigen continues to make improvements and advances in apoptosis KITS detection with our newest method for labeling cells and tissues. It is based on a modified technique that incorporates the sensitivity of Trevigen’s traditional TUNEL-based tissue-specific kits, but with the added advantage of enhanced signal-to-noise ratio. 125 Specialty In Situ Apoptosis Detection Kits

C a t a l o g # D e s c r i p t i o n Light MicroscopyFluorescent Flow Microscopy CytometryGel Electrophoresis Plate ReaderDouble LabelConfocal UnfixedMicroscopy CellsFixed CellsParaffin EmbeddedFrozen TissueFresh Tissue TissuePage 4827-30-K CardioTACS™ 126 4829-30-K DermaTACS™ 128 4823-30-K NeuroTACS™ II 130 4815-30-K TumorTACS™ 132 4826-30-K VasoTACS™ 134

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APOPTOSIS DETECTION

DNA Fragmentation CardioTACS™ In Situ Apoptosis Detection Kit

The CardioTACS™ Kit was developed to provide the heart researcher with an effective method for identifying apoptotic cells in cardiac samples. The high cellularity of cardiac tissue presents problems in permeabilization, so the CardioTACS™ Kit comes with two permeabilization reagents to provide options. The kit is based on DNA end-labeling using terminal deoxynucleotidyl transferase (TdT) and a modified nucleotide that is subsequently detected using our TACS Blue Label™ detection system. Trevigen has developed an exclusive apoptosis grade TdT enzyme when coupled with the TACS Blue Label™ solution, provides labeling 20 to 50 times more sensitive than standard diaminobenzidine (DAB) labeling methods.To ensure ease of data interpretation when the numbers of apoptotic cells are low, CardioTACS™ includes Nuclear Fast Red Counterstain which provides superb contrast to the TACS Blue Label™ in apoptotic cells. In addition, the kit includes TACS-Nuclease™ that is used to generate a positive control in your own sample. The positive control provides an internal control for permeabilization and labeling so additional optimization is nominal and the data can be Apoptotic rat cardiac myocyte labeled using the CardioTACS™ Kit. Rat heart tissue was fixed in 4% interpreted with confidence. paraformaldehyde overnight followed by paraffin embedding. Five micron sections were prepared and placed onto glass This complete kit provides all the reagents required for labeling including two permeabilization microscope slides. The sample was processed following the reagents, labeling and detection reagents, stop buffer, counterstain, TACS-Nuclease™ reagent, CardioTACS™ Kit protocol. Photo courtesy Dr. J. Zhang, FDA. and a protocol detailing incubation times and reagent concentrations.

FEATURES:

CardioTACS™ • Fast. Requires less than 3 hours to complete TUNEL assay. • Exclusive, non-toxic TACS Safe™ TdT buffer - sodium cacodylate free. 126 • Unique buffer system produces more consistent labeling. • Performance tested on heart-derived samples. • Includes exclusive Cytonin™ permeabilization reagent. • Includes TACS-Nuclease™ solution for preparing sample-dependent positive controls.

APPLICATIONS: • In situ detection of apoptosis in fixed frozen, paraffin embedded, or plastic embedded cardiac cells and tissues. • Assists in the identification of apoptotic morphologies. • Helps resolve unique problems encountered when detecting apoptotic cardiac cells.

COMPONENTS: Catalog # Component Size 4800-30-01 Proteinase K 30 µl 4800-30-06 Strep-HRP 30 µl 4800-30-11 TACS Blue Label™ 3 ml 4800-30-12 Blue Strep-HRP Diluent 7.5 ml 4800-30-15 TACS-Nuclease™ 15 µl 4800-30-16 TACS-Nuclease Buffer 1.5 ml 4800-30-17 Nuclear Fast Red 50 ml 4810-30-02 TACS® 2 TdT Labeling Buffer 100 ml 4810-30-03 TACS® 2 TdT Stop Buffer 100 ml 4810-30-04 TACS® 2 TdT dNTP 30 µl 4810-30-05 TdT Enzyme 30 µl 4810-30-14 Manganese Cation (Sold as 4810-90-14) 30 µl 4876-05-01 Cytonin™ 5 ml

ORDERING INFORMATION

4827-30-K CardioTACS™ Kit, 30 Samples

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APOPTOSIS DETECTION

DNA Fragmentation CardioTACS™ In Situ Apoptosis Detection Kit

ITEMS NOT INCLUDED:

Reagents for dehydration and rehydration of paraffin embedded sections, H2O2, methanol, PBS buffer, mounting medium, and disposables.

RELATED PRODUCTS: • PARP/Apoptosis Assays • Apoptosis Antibodies • TACS™ Apoptotic DNA Laddering Kit • TACS® 2 Replenisher Kit • Cell Proliferation Assays

STORAGE: Store components at -20˚C, 4˚C, and room temperature.

REFERENCES: CardioTACS™ 1. Gavrieli, Y., Y. Sherman, and S.A. Ben-Sasson. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell. Biol. 119:493-501. 2. Lovelace, C.I.P., J. Zhang, P.G. Vanek, and G.B. Collier. 1996. Detecting apoptotic cells in situ. Biomedical Products 21:76-77. 3. Thiry, M. 1992. Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues. J. Histochem. Cytochem. 40:411-419. 4. Bueno, O., L. DeWindt, K. Tymitz, S. Witt, T. Kimball, R. Klevitsky, T. Hewett, S. Jones, D. 127 Lefer, C. Peng, R. Kitsis, and J. Molkentin. 2000. The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO. J. 19:6341-6350.

PRODUCT CITATION: 1. Liu, X., J. A. Simpson, K.R. Brunt, C. A. Ward, S.R.R. Hall, R.T. Kinobe, V. Barrette, M.Y. Tse, S.C. Pang, A.S. Pachori, V.J. Dzau, K.O. Ogunyankin, and L.G. Melo. 2007. Preemptive heme oxygenase-1 gene delivery reveals reduced mortality and preservation of left ventricular function 1 yr after acute myocardial infarction. Am. J. Physiol. Heart. Circ. Physiol. 293: H48 - H59.

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APOPTOSIS DETECTION

DNA Fragmentation DermaTACS™ In Situ Apoptosis Detection Kit

The DermaTACS™ Kit was developed to provide researchers with an effective method for measuring apoptosis in skin samples. The kit was based on DNA end-labeling using terminal deoxynucleotidyl transferase (TdT) and modified nucleotides. Detection of incorporated molecules is achieved using a chromogenic substrate with a Streptavidin horseradish peroxi- 1 hour post UVB dase detection system. The extracellular scaffolding in skin tissue can make it problematic to permeabilize and high background is a common problem with skin samples. The DermaTACS™ Kit includes two permeabilization reagents as options for permeabilization along with a detailed protocol with hints and tips for optimal labeling of skin samples based on empirical testing. Low background is achieved using a combination of the high specificity of Trevigen’s unique 6 hour post UVB cation-based optimized TdT labeling reaction using Apoptosis Grade™ TdT and TACS Blue Label™ as the visualization method. Also included is TACS-Nuclease™ to generate a positive control from your own sample. In conjunction with the excellent contrast between the TACS Blue Label™ and the Red Counterstain C, the positive control ensures easy data interpretation.

TACS-Nuclease™ Positive Control This complete kit provides all the reagents required for labeling including two permeabilization Detection of DNA fragmentation in UVB irradiated human reagents, labeling and detection reagents, stop buffer, counterstain, and TACS-Nuclease™. skin model, EpiDerm™, with Trevigen’s in situ apoptosis detection kit for skin cells and tissues, DermaTACS™. FEATURES: The dark blue stained cells at 1 and 6 hours also exhibit • Fast. Requires less than 3 hours to complete. punctate morphology indicative of apoptosis. The TACS-Nuclease™ treated sample shows the diffuse blue • Exclusive, non-toxic TACS Safe™ TdT buffer - sodium cacodylate free.

DermaTACS™ staining of fragmented but uncondensed DNA. Samples • Unique buffer system produces more consistent labeling. were provided courtesy of Dr. Patrick Hayden, MatTek • Performance tested on skin-derived samples. 128 Corporation, Ashland, MA. • Includes exclusive Cytonin™ permeabilization reagent. • Includes TACS-Nuclease™ solution for preparing sample-dependent positive controls.

APPLICATIONS: • In situ detection of apoptosis (by TUNEL) in fixed frozen, paraffin embedded, or plastic embedded cells and tissue sections. • Assists in the identification of apoptotic morphologies. • Helps resolve unique problems encountered when detecting apoptosis in skin sections.

COMPONENTS: Catalog # Component Size 4800-30-01 Proteinase K 30 µl 4800-30-06 Strep-HRP 30 µl 4800-30-11 TACS Blue Label™ 3 ml 4800-30-15 TACS-Nuclease™ 15 µl 4800-30-16 TACS-Nuclease Buffer 1.5 ml 4800-30-19 Red Counterstain C 50 ml 4810-30-02 TACS 2 TdT Labeling Buffer 100 ml 4810-30-03 TACS 2 TdT Stop Buffer 100 ml 4810-30-05 TdT Enzyme 30 µl 4828-30-04 TACS B-dNTP Mix 30 µl 4828-30-06 Anti-BrdU antibody 30 µl 4828-30-12 Strep-Diluent 7.5 ml 4876-05-01 Cytonin™ 5 ml

ORDERING INFORMATION

4829-30-K DermaTACS™ Kit, 30 Samples 4800-30-42 EpiDerm™ Control Slides, 2 slides

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APOPTOSIS DETECTION

DNA Fragmentation DermaTACS™ In Situ Apoptosis Detection Kit

ITEMS NOT INCLUDED:

Reagents for dehydration and rehydration of paraffin embedded sections, H2O2, methanol, PBS buffer, mounting medium, and disposables.

RELATED PRODUCTS: • EpiDerm™ Control Slides • TACS™ DNA Laddering Kit • TACS•XL® Replenisher Kit • PARP/Apoptosis Assays • Cell Proliferation Assays

STORAGE: Components are stored at -20˚C, 4˚C, and room temperature.

CONTROLS: DermaTACS™ EpiDerm™ Control Slides (Catalog # 4800-30-42) available separately.

PRODUCT CITATION: 1. Zagon, I.S., M.S. Klocek, J.W. Sassani, and P.J. McLaughlin. 2007. Use of topical insulin to normalize corneal epithelial healing in diabetes mellitus. Arch. Ophthalmol. 125:1082-1088. 2. Bedelbaeva, K., A. Snydera, D. Gourevitcha, L. Clarka, X-M. Zhanga, J. Leferovicha, J.M. Cheverudb, P. Liebermana, and E. Heber-Katza. 2010. Lack of p21 expression links cell cycle control and appendage regeneration in mice. Proc. Natl. Acad. Sci. U.S.A. 107:5845-5850. 3. Khanna S, S. Biswas, Y. Shang, E. Collard, A. Azad, C. Kauh, V. Bhasker, G.M. Gordillo, C.K. Sen, and S. Roy. 2010. Macrophage dysfunction impairs resolution of inflammation in the wounds of diabetic mice. PLoS One. 5:e9539.

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APOPTOSIS DETECTION

DNA Fragmentation NeuroTACS™ II In Situ Apoptosis Detection Kit

NeuroTACS™ II is a complete reagent kit optimized to provide rapid and convenient identification of apoptosis in brain tissue or neuronal cells. The kit has been developed to overcome the common difficulties unique to neuronal samples including the fragile nature of brain tissue sections, high background problems, poor counterstaining with common dyes, and the need to perform dual labeling experiments to detect cell specific antigens in conjunction with apoptotic cells. A key feature is NeuroPore™, a proprietary permeabilization reagent that gently permeabilizes samples while retaining cell morphology. NeuroPore™ also contains blocking reagents to allow its use as an antibody diluent in immunohistochemistry and to reduce background staining. DNA fragments generated by apoptosis are end-labeled with modified nucleotides using a highly purified terminal deoxynucleotidyl transferase enzyme (TdT). The incorporated nucleotides are detected using a horseradish peroxidase system that catalyzes the conversion of diaminobenzidine (DAB) into a visible dark brown precipitate. NeuroTACS™ II contains the Blue Counterstain to allow visualization of all cells within the sample with good contrast to the brown DAB precipitate. A DAB enhancer is also provided with the kit for the option to intensify or darken DAB staining. In addition, the Blue Counterstain is compatible with the Double labeling of mouse brain section for apoptosis using Neuro- red-colored substrates used for phosphatase detection in double labeling experiments. The protocol TACS™ II (brown) and a monoclonal antibody to GFAP (red). Brain includes details for labeling in situ for apoptosis and antigen detection on the same sample. sections were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 µM. The section was counterstained To ensure that your own samples have been processed and permeabilized correctly, we provide using Trevigen’s Blue Counterstain. TACS-Nuclease™, a unique reagent used to generate a positive control with your own samples. This provides a high degree of confidence for data interpretation and can help pinpoint problem steps in the labeling procedure. NeuroTACS™ II NeuroTACS™

FEATURES: 130 • Fast. Requires less than 3 hours to complete. • Exclusive, non-toxic TACS Safe™ TdT buffer - sodium cacodylate free. • Unique buffer system produces more consistent labeling. • Performance tested on brain sections. • Includes exclusive NeuroPore™ permeabilization reagent. • Includes TACS-Nuclease™ solution for preparing sample-dependent positive controls.

APPLICATIONS: • In situ detection of apoptosis (by TUNEL) in fixed frozen, paraffin embedded, or plastic embedded cells and tissues. • Assists in the identification of apoptotic morphologies. • Helps resolve unique problems encountered when detecting apoptotic neuronal cells.

COMPONENTS: Catalog # Component Size 4800-30-01 Proteinase K 30 µl 4800-30-06 Strep-HRP 30 µl 4800-30-07 Diaminobenzidine 3.75 ml 4800-30-09 DAB Enhancer Reagent 1 ml 4800-30-15 TACS-Nuclease™ 15 µl 4800-30-16 TACS-Nuclease Buffer 1.5 ml 4810-30-02 TACS 2 TdT Labeling Buffer 100 ml 4810-30-03 TACS 2 TdT Stop Buffer 100 ml 4810-30-04 TACS 2 TdT dNTP 30 µl 4810-30-05 TdT Enzyme 30 µl 4810-30-14 Manganese Cation (Sold as 4810-90-14) 30 µl 4820-30-01 NeuroPore™ 5 ml 4820-30-13 Blue Counterstain 50 ml

ORDERING INFORMATION

4823-30-K NeuroTACS™ II Kit, 30 Samples

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APOPTOSIS DETECTION

DNA Fragmentation NeuroTACS™ II In Situ Apoptosis Detection Kit

ITEMS NOT INCLUDED:

Reagents for dehydration and rehydration of paraffin embedded sections, H2O2, methanol, PBS buffer, mounting medium, and disposables.

RELATED PRODUCTS: • TACS™ Apoptotic DNA Laddering Kit • TACS® 2 Replenisher Kit • PARP/Apoptosis Assays • Cell Proliferation Assays

STORAGE: Components are stored at -20˚C, 4˚C, and room temperature.

REFERENCES: 1. Gavrieli, Y., Y. Sherman, and S.A. Ben-Sasson. 1992. Identification of programmed cell death NeuroTACS™ II in situ via specific labeling of nuclear DNA fragmentation. J. Cell. Biol. 119:493-501. 2. Lovelace, C.I.P., J. Zhang, P.G. Vanek, and G.B. Collier. 1996. Detecting apoptotic cells in situ. Biomedical Products 21:76-77. 3. Thiry, M. 1992. Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues. J. Histochem. Cytochem. 40:411-419.

PRODUCT CITATION: 131 1. Samuel, M.A., J.D. Morrey, and M.S. Diamond. 2007. Caspase 3-Dependent Cell Death of Neurons Contributes to the Pathogenesis of West Nile Virus Encephalitis J. Virol. 81: 2614 - 2623.

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APOPTOSIS DETECTION

DNA Fragmentation TumorTACS™ In Situ Apoptosis Detection Kit

TumorTACS™ is a complete reagent kit providing rapid and convenient identification of apoptosis in tumors or cancer cells. The labeling of tumor-containing specimens can be problematic due to the high levels of necrosis in these tissues. During necrosis, cessation of homeostasis, cell swelling, and nuclear membrane rupture generates highly degraded DNA that can interfere with interpretation of data. Trevigen’s kit has an optimized TUNEL-based system that preferentially labels the double-stranded DNA found in apoptotic cells. DNA fragments generated during apoptosis are end-labeled with modified nucleotides using a highly purified terminal deoxynucleotidyl transferase enzyme (TdT) in a unique non-toxic labeling buffer supplemented with cations for apoptosis-specific labeling. The incorporated nucleotides are subsequently detected using a horseradish peroxidase conjugate. The conjugate catalyzes the conversion of diaminobenzidine (DAB) into a visible dark brown precipitate. The kit includes methyl green counterstain which is a general counterstain appropriate for a wide variety of tissues and tumor types and provides good contrast with the brown DAB in labeled cells.

Apoptotic cells within mouse mammary tumor identified A DAB enhancer is also provided with the kit for the option to intensify or darken DAB staining. using the TumorTACS™ kit. Mammary tumor was fixed in TumorTACS™ contains all the reagents required for permeabilizing, labeling, staining, and 4% paraformaldehyde overnight followed by paraffin counterstaining tumor samples, including TACS-Nuclease™ for preparing positive controls. embedding. Five micron sections were prepared and placed onto glass microscope slides. The sample was FEATURES: processed following the TumorTACS™ kit protocol. • Fast. Requires less than 3 hours to complete. • Exclusive, non-toxic TACS Safe™ TdT buffer - sodium cacodylate free. • Unique buffer system produces more consistent labeling. TumorTACS™ • Performance tested on tumor samples. • Includes exclusive Cytonin™ permeabilization reagent. 132 • Includes TACS-Nuclease™ solution for preparing sample-dependent positive controls.

COMPONENTS: Catalog # Component Size 4876-05-01 CytoninTM 5 ml 4800-30-01 Proteinase K 30 µl 4810-30-02 TACS 2 TdT Labeling Buffer 100 ml 4810-30-03 TACS 2 TdT Stop Buffer 100 ml 4810-30-04 TACS 2 TdT dNTP 30 µl 4810-30-05 TdT Enzyme 30 µl 4810-30-14 Manganese Cation (Sold as 4810-90-14) 30 µl 4800-30-06 Strep-HRP 30 µl 4800-30-07 Diaminobenzidine 3.75 ml 4800-30-09 DAB Enhancer Reagent 1 ml 4800-30-15 TACS-Nuclease™ 15 µl 4800-30-16 TACS-Nuclease Buffer 1.5 ml 4800-30-18 1% Methyl Green 50 ml

ORDERING INFORMATION

4815-30-K TumorTACS™ Kit, 30 Samples

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APOPTOSIS DETECTION

DNA Fragmentation TumorTACS™ In Situ Apoptosis Detection Kit

ITEMS NOT INCLUDED:

Reagents for dehydration and rehydration of paraffin embedded sections, H2O2, methanol, PBS buffer, mounting medium, and disposables.

RELATED PRODUCTS: • TACS™ Apoptotic DNA Laddering Kit • TACS® 2 Replenisher Kit • PARP/Apoptosis Assays • Cell Proliferation Assays

STORAGE: Components are stored at -20˚C, 4˚C and room temperature.

REFERENCES:

1. Gavrieli, Y., Y. Sherman, and S.A. Ben-Sasson. 1992. Identification of programmed cell death TumorTACS™ in situ via specific labeling of nuclear DNA fragmentation. J. Cell. Biol. 119:493-501. 2. Lovelace, C.I.P., J. Zhang, P.G. Vanek, and G.B. Collier. 1996. Detecting apoptotic cells in situ. Biomedical Products 21:76-77. 3. Thiry, M. 1992. Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues. J. Histochem. Cytochem. 40:411-419.

PRODUCT CITATION: 133 1. Kawakubo, T., K. Okamoto, J. Iwata, M. Shin, Y. Okamoto, A. Yasukochi, K.I. Nakayama, T. Kadowaki, T. Tsukuba, and K. Yamamoto. 2007 Cathepsin E Prevents Tumor Growth and Metastasis by Catalyzing the Proteolytic Release of Soluble TRAIL from Tumor Cell Surface. Cancer Res. 67:10869–10878.

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APOPTOSIS DETECTION

DNA Fragmentation VasoTACS™ In Situ Apoptosis Detection Kit

The VasoTACS™ Kit was developed to provide an effective method for identifying apoptotic cells in vascular samples. This kit allows the user to successfully label apoptotic cells, including endothelial and smooth muscle cells, throughout the vascular system. Similar to the CardioTACS™ Kit, this kit is also based on DNA end-labeling using terminal deoxynucleotidyl transferase (TdT) and a modified nucleotide that is subsequently detected using our TACS Blue Label™ detection system with Red Counterstain C. Specifically, VasoTACS™ has been tested and optimized in order to help eliminate background and improve labeling in vascular tissue. FEATURES: • Fast. Requires less than 3 hours to complete. • Exclusive, non-toxic TACS Safe™ TdT buffer - sodium cacodylate free. Drug-induced apoptosis in the small artery of a rat • Unique buffer system produces more consistent labeling. exhibiting spontaneous hypertension using the Trevigen • Performance tested on vascular tissues. VasoTACS™ Kit. The tissue was formalin-fixed and • Includes exclusive Cytonin™ permeabilization reagent. paraffin-embedded. Photo courtesy of Dr. Jun Zhang, FDA. APPLICATIONS: • In situ detection of apoptosis (by TUNEL) in fixed frozen, paraffin embedded, or plastic embedded vascular cells and tissues. • Assists in the identification of apoptotic morphologies. • Helps resolve unique problems encountered when detecting apoptotic vascular cells.

COMPONENTS: VasoTACS™ Catalog # Component Size 4800-30-01 Proteinase K 30 µl 134 4800-30-06 Strep-HRP 30 µl 4800-30-11 TACS Blue Label™ 3 ml 4800-30-12 Blue Strep-HRP Diluent 7.5 ml 4800-30-15 TACS-Nuclease™ 15 µl 4800-30-16 TACS-Nuclease Buffer 1.5 ml 4800-30-19 Red Counterstain C 50 ml 4810-30-02 TACS 2 TdT Labeling Buffer 100 ml 4810-30-03 TACS 2 TdT Stop Buffer 100 ml 4810-30-04 TACS 2 TdT dNTP 30 µl 4810-30-05 TdT Enzyme 30 µl 4810-30-14 Manganese Cation (Sold as 4810-90-14) 30 µl 4876-05-01 Cytonin™ 5 ml

ITEMS NOT INCLUDED:

Reagents for dehydration and rehydration of paraffin embedded sections, H2O2, methanol, PBS buffer, mounting medium, and disposables.

RELATED PRODUCTS: • TACS™ Apoptotic Laddering Kit • TACS® 2 Replenisher Kit • PARP/Apoptosis Assays • Cell Proliferation Assays

STORAGE: Components are stored at -20˚C, 4˚C and room temperature.

REFERENCES: 1. Gavrieli, Y., Y. Sherman, and S.A. Ben-Sasson. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell. Biol. 119:493-501. ORDERING INFORMATION 2. Lovelace, C.I.P., J. Zhang, P.G. Vanek, and G.B. Collier. 1996. Detecting apoptotic cells in situ. Biomedical Products 21:76-77. 4826-30-K VasoTACS™ Kit, 30 Samples PRODUCT CITATION: 1. Skelly, C.L., A. Chandiwal, J.E. Vosicky, R.R. Weichselbaum, and B. Roizman 2007. Attenuated herpes simplex virus 1 blocks arterial apoptosis and intimal hyperplasia induced by balloon 1-800-873-8443 • www.trevigen.com angioplasty and reduced blood flow. Proc. Natl. Acad. Sci, 104:12474-12478. INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:21 PM Page 139

APOPTOSIS DETECTION

DNA Fragmentation TiterTACS™ Colorimetric Apoptosis Detection Kit

The TiterTACS™ Colorimetric Apoptosis Detection Kit takes advantage of Trevigen’s exclusive in situ labeling technology bringing it to the 96 well microplate format for high throughput quantitative detection of apoptosis. Detection using TACS-Sapphire™, a non-toxic colorimetric substrate, allows both kinetic and endpoint readings. The reaction generates a blue product that can be measured at 630 nm, and will turn yellow after the reaction is stopped with acid allowing endpoint reading at 450 nm.

During the process of apoptosis, DNA fragmentation occurs following the activation of endonucleases. The labeling of the 3’ ends of DNA fragments provides an easy measure of cells undergoing apoptosis. Modified nucleotides are incorporated at the 3’ ends by the activity of terminal deoxynucleotidyl transferase (TdT). These nucleotides are detected using Detection of apoptosis in ML-1 cells after treatment with 1 µM staurosporine. All control wells contained 1x105 cells. Cells a horseradish-peroxidase detection system and TACS-Sapphire™. TiterTACS™ can be used were harvested, fixed and labeled according to the TiterTACS™ with suspension or monolayer cells. The kit is designed to use fixed samples, allowing you protocol prior to colorimetric analysis. Reaction was stopped to work safely with samples that are infected with biohazardous agents. Fixed samples may with 2N HCl. be stored conveniently during time-course experiments.

The TiterTACS™ Kit also provides TACS-Nuclease™ solution to generate positive controls from TiterTACS™ your own samples giving you a maximal value for the assay.

FEATURES: • Fast. Requires less than 4 hours to complete. • Exclusive, non-toxic TACS Safe™ TdT buffer - sodium cacodylate free. • Unique buffer system produces more consistent labeling. • Includes exclusive Cytonin™ permeabilization reagent. 135 • Includes TACS-Nuclease solution for preparing sample-dependent positive controls. • Convenient, 96 well microplate format.

APPLICATIONS: • Identification and quantitation of apoptosis in cultured cells (by TUNEL).

COMPONENTS: Catalog # Component Size 4876-60-01 CytoninTM 6 ml 4821-96-01 Proteinase K 100 µl 4817-60-02 TACS 2 TdT Labeling Buffer 20 ml 4817-60-03 TACS 2 TdT Stop Buffer 20 ml 4821-96-04 TdT dNTP Mix 35 µl 4821-96-05 TdT Enzyme 35 µl 4821-96-14 50X Mn2+ 100 µl 4800-30-06 Strep-HRP 30 µl 4800-30-12 Blue-Strep Diluent 7.5 ml 4822-96-08 TACS-Sapphire™ 10 ml 4800-30-15 TACS-Nuclease™ 15 µl 4800-30-16 TACS-Nuclease Buffer 1.5 ml

ITEMS NOT INCLUDED:

Reagents for dehydration and rehydration, H2O2, methanol, PBS buffer, and disposables. Centrifuge equipped with microplate adapters.

RELATED PRODUCTS: • TACS™ Apoptotic DNA Laddering Kit • FlowTACS™ • TACS™ Replenisher Kit ORDERING INFORMATION • PARP/Apoptosis Assays • Cell Proliferation Assays 4822-96-K TiterTACS™ Colorimetric, 96 Wells

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DNA Fragmentation TiterTACS™ Colorimetric Apoptosis Detection Kit

STORAGE: Components are stored at -20˚C, 4˚C, and room temperature.

REFERENCES: 1. Negoescu, A., P. Lorimier, F. Labat-Moleur, C. Drouet, C. Robert, C. Guillermet, C. Brambilla, and E. Brambilla. 1996. In situ apoptotic cell labeling by the TUNEL method: improvements and evaluation on cell preparation. J. Histochem. Cytochem. 44:959-68. 2. Kerr, J.F., G.C. Gobe, C.M. Winterford, and B.V. Harmon. 1995. Anatomical methods in cell death. Methods Cell. Biol. 46:1-27. 3. Tomei, L.D. and F.O. Cope (ed). 1994. Apoptosis II: The Molecular Basis of Apoptosis in Disease. Current Communications in Cell and Molecular Biology, Vol 8. New York:Cold Spring Harbor Laboratory Press. 4. Yamawaki, M, A. Zurbriggen, A. Richard, and M. Vandevelde. 1993. Saponin treatment for in situ hybridization maintains good morphological preservation. J. Histochem. Cytochem. 41:105-109. 5. Tomei, L.D. and F.O. Cope (ed). 1991. Apoptosis: The Molecular Basis of Cell Death. Current Communications in Cell and Molecular Biology, Vol 3. New York:Cold Spring Harbor Laboratory Press. 6. Thiry, M. 1992. Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues. J. Histochem.

TiterTACS™ Cytochem. 40:411-419.

136 PRODUCT CITATION: 1. Soule, B.P., J.M. Brown, N.M. Kushnir-Sukhov, N.L. Simone, J.B. Mitchell, and D.D. Metcalfe. 2007. Effects of gamma radiation on FcRI and TLR-mediated mast cell activation J. Immunol. 179:3276-3286. 2. Lee C.S., Y.J. Kim, E.R. Jang, S.C. Myung, and W. Kim. Akt inhibitor enhances apoptotic effect of carboplatin on human epithelial ovarian carcinoma cell lines. Eur J Pharmacol. 632:7-13

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DNA Fragmentation FlowTACS™ Apoptosis Detection Kit

FlowTACS™ takes advantage of Trevigen’s exclusive in situ labeling technology to label cell samples for processing by flow cytometry. FlowTACS™ also provides flexibility in selection of fluorophores (see page 158) that are compatible with your research design. The kit allows multi-color labeling in conjunction with experiment-specific antibodies.

DNA fragmentation is an irreversible, committed step in apoptosis, and the labeling of 3’ ends provides an easy measure of cells undergoing apoptosis. Cells may also be analyzed for DNA content using the included propidium iodide/RNase A solution. The FlowTACS™ Kit also provides TACS-Nuclease™ to generate positive controls for calibrating your flow cytometer. The FlowTACS™ Kit uses fixed cells, allowing you to safely work with cells that are infected with biohazardous agents. Also, samples may be stored conveniently during time-course experiments.

This complete kit provides all the reagents required for labeling including two permeabilization reagents, labeling and stop buffers, labeling and detection reagents, and TACS-Nuclease™ for generating positive controls with your own samples. Please refer to our components FlowTACS™ listing for details. A FEATURES: • Fast. Requires less than 3 hours to complete. • Exclusive, non-toxic TACS Safe TdT™ buffer - sodium cacodylate free. • Unique buffer system produces more consistent labeling. • Works on fixed cells. 137 • Includes exclusive Cytonin™ permeabilization reagent. • Includes TACS-Nuclease™ solution for preparing sample-dependent positive controls.

APPLICATIONS: • Identify and quantitate cultured apoptotic cells (by TUNEL and Flow Cytometry). • Bi-color and tri-color analysis.

COMPONENTS: Catalog # Component Size 4876-60-01 Cytonin™ 6 ml 4817-60-02 TACS 2 TdT Labeling Buffer 20 ml 4817-60-03 TACS 2 TdT Stop Buffer 20 ml 4810-30-04 TdT dNTP Mix 30 µl B 4810-30-05 TdT Enzyme 30 µl 4810-30-14 50X Mn2+ 100 µl µ Analysis of murine thymocytes at 16 hours after treatment with 10 µg/ml 4800-30-14 Strep-Fluorescein 30 l cycloheximide (A) and 1 µM dexamethasone (B). Cells were harvested 4817-60-04 Propidium Iodide/RNAse 1 ml and labeled according to the FlowTACS™ protocol prior to analysis by 4800-30-15 TACS-Nuclease™ 15 µl flow cytometry. Data courtesy N. Hardegen, NIH, NIDR, Bethesda, MD. 4800-30-16 TACS-Nuclease Buffer 1.5 ml

RELATED PRODUCTS: • TiterTACS™ • TACS™ Annexin V Kits • DePsipher™ Mitochondrial Potential Assay • MitoShiftTM Mitochondrial Potential Assay • TACS™ Apoptotic DNA Laddering Kit • TACS™ Replenisher Kit ORDERING INFORMATION • Cell Proliferation Assay

4817-60-K FlowTACS™ Kit, 60 Tests

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DNA Fragmentation FlowTACS™ Apoptosis Detection Kit

ITEMS NOT INCLUDED:

Reagents for dehydration and rehydration, H2O2, methanol, PBS buffer, and disposables.

STORAGE: Store components at -20˚C, 4˚C, and room temperature.

REFERENCES: 1. Ormerod, M.G. (ed). 1994. Flow Cytometry: a practical approach, 2nd edition. New York:IRL Oxford University Press, Inc. 2. Negoescu, A., P. Lorimier, F. Labat-Moleur, C. Drouet, C. Robert, C. Guillermet, C. Brambilla, and E. Brambilla. 1996. In situ apoptotic cell labeling by the TUNEL method: improvements and evaluation on cell preparation. J. Histochem. Cytochem. 44:959-968. 3. Shi, S.-R., R.J. Cote, and C.R. Taylor. 1997. Antigen retrieval immunocytochemistry: practice and development. J. Histotech. 20:145-154. 4. Thiry, M. 1992. Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues. J. Histochem. Cytochem. 40:411-419.

PRODUCT CITATION: 1. Wang L., T.C. Schulz, E.S. Sherrer, D.S. Dauphin, S. Shin, A.M. Nelson, C.B. Ware, M.Zahn, C-Z.

FlowTACS™ Song, X. Chen, S.N. Brimble, A.McLean, M.J. Galeano, E.W. Uhl, K.A. D’Amour, J.D. Chesnut, M.S. Rao, A. Blau, and A.J. Robins. 2007 Self-renewal of human embryonic stem cells requires 138 insulin-like growth factor-1 receptor and ERBB2 receptor signaling Blood, 110:4111-4119. 2. Gupta M.K., R.S. Papay, C.W. Jurgens, R.J. Gaivin, T. Shi, V.A. Doze, and D.M. Perez. 2009. alpha1-Adrenergic receptors regulate neurogenesis and gliogenesis. Mol Pharmacol. 76:314-26.

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DNA Fragmentation TACS® Apoptotic DNA Laddering Kit

The TACS® Apoptotic DNA Laddering Kit is used to detect and estimate the level of internu- cleosomal DNA fragmentation that occurs during apoptosis. Evidence of DNA laddering supports other experimental data including positive Annexin V staining, PARP cleavage and morphological identification methods to verify that apoptosis occurred. The kit contains all reagents necessary to isolate, label, and detect DNA.

For those researchers investigating apoptosis in tissues, a supplemental Tissue Extraction Kit is available. This kit provides the reagents necessary to prepare tissues for DNA extraction.

STORAGE: Store components at -20˚C, 4˚C, and room temperature. DNA LADDERING KIT COMPONENTS: DNA Laddering kit with DNA from control and apoptotic cells. DNA was isolated from WEHI cells that were untreated (A) Catalog # Component Size or treated 50 µM Etoposide overnight (B). Two µg of DNA 4850-20-01 Lysis Solution 1 2 x 1 ml was displayed on a 1.5% TreviGel™ 500 gel containing 4850-20-02 Extraction Solution 2 20 ml ethidium bromide and subjected to electrophoresis at 8 V/cm 4850-20-03 Extraction Buffer 3 8 ml for 1 hour. 4850-20-04 Sodium Acetate 4 1 ml 4850-20-05 DNase-Free Water 5 2 ml 4850-20-10 5X Gel Loading 250 µl 9804-006-P TreviGel™ 500 6 gm 4859-20-01* 10X Tissue Buffer 500 µl 4850-20-06 Sample Buffer 6 2 ml 139 *Additional reagent required for extracting DNA from tissue

RELATED PRODUCTS: • TreviGel™ (see page 182) • TACS™ Genomic DNA isolation Kit • Annexin V Kits • PARP/Apoptosis Assays

REFERENCES: 1. Rosl, F. 1992. A simple method for detection of apoptosis in human cells. Nucleic Acid Res. 201:5243. 2. Smith, M.L. and A.J. Fornace. 1996. Mammalian DNA damage-inducible genes associated with growth arrest and apoptosis. Mutat. Res. 340:109-124.

PRODUCT CITATION: 1. Inoue H., N. Sameshima, T. Ishida, A. Tsuji, K. Kudo, and N. Ikeda. 2006. Vulnerability of experimentally induced fatty liver to heat stress in rats. J. Gastroenterol. 41:55-61. 2. Yoo DR, Jang YH, Jeon YK, Kim JY, Jeon W, Choi YJ, Nam MJ. 2009. Proteomic identification of anti-cancer proteins in luteolin-treated human hepatoma Huh-7 cells. Cancer Lett. 282:48-54.

ORDERING INFORMATION

4850-20-ET Ethidium Bromide DNA Laddering Kit, 20 Wells 4859-20-K Tissue Extraction Reagents, 20 Wells

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Mitochondrial Events DePsipher™ Mitochondrial Potential Assay

In the intrinsic apoptosis pathway, mitochondrial permeability transition is an important event ΔΨ wherein the electrochemical gradient (referred to as delta-psi or m) across the mitochondrial membrane collapses. This collapse occurs through the formation of channels or pores in the outer mitochondrial membrane, which involves Bax insertion and oligomerization, followed by the release of Cytochrome C into the cytoplasm.

The DePsipher™ Kit uses a unique cationic dye (5,5’6,6’-tetrachloro-1,1’,3, 3’-tetraethylbenzimida- ΔΨ zolyl-carbocyanine iodide) to indicate the loss of m. The dye readily enters cells and fluoresces brightly red in its multimeric form within healthy mitochondria. In apoptotic cells, the mitochondrial membrane potential collapses, and the DePsipher™ reagent cannot accumulate within the Identification of apoptotic cells using DePsipher™. INT407 human cells were treated with 25 µM etoposide for 8 mitochondria. In these cells, DePsipher™ returns to its green fluorescent monomeric form. Apoptotic hours, and treated with the DePsipher™ reagent in Reaction cells, showing primarily green fluorescence, are thus easily differentiated from healthy cells which Buffer for 30 minutes prior to visualization. Healthy cells show red fluorescence. The aggregate red form has absorption/emission maxima of 585/590 nm, (containing red aggregates) can be differentiated from and the green monomeric form has absorption/emission maxima of 510/527 nm. Both apoptotic and apoptotic cells (containing mostly green monomers). healthy cells can be visualized simultaneously by epifluorescence microscopy using a wide band-pass filter.

The DePsipher™ reagent is easy to use. Simply resuspend the reagent in Reaction Buffer or culture media (with or without the Stabilizer Solution), add to your cells, incubate for 15 to 20 minutes, wash and analyze by flow cytometry or microscopy. Visualization by microscopy allows a rapid

DePsipher™ inspection and qualification of apoptosis. Flow cytometric analysis allows easy quantitation of cell death as evidenced by mitochondrial potential breakdown. 140 FEATURES: • Simple. Just add DePsipher™ reagent to media or reaction buffer. • Unique Stabilizer Solution improves results. • Fast. Takes only 20 minutes. • Flexible. View cells by epifluorescence or confocal microscopy, or analyze cells by flow cytometry.

APPLICATIONS: • Flow cytometry • Epifluorescence microscopy • Confocal microscopy

A B

Rat thymocytes analyzed using DePsipher™ by flow cytometry. Cells were collected after 24 hours following either no treatment (A), ORDERING INFORMATION or treatment with 1 µM dexamethasone (B). Cells were incubated with the DePsipher™ reagent in 1X Reaction Buffer for 20 minutes, washed, and analyzed by flow cytometry. 6300-100-01 DePsipher™ (2.5 mg/ml), 100 µl 6300-100-K DePsipher™ Kit, 100 Tests

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APOPTOSIS DETECTION

Mitochondrial Events DePsipher™ Mitochondrial Potential Assay

COMPONENTS: Catalog # Component Size 6300-100-01 DePsipher™, 2.5 mg/ml 100 µl 6300-100-02 10X Reaction Buffer 2 x 30 ml 6300-100-03 Stabilizer Solution 5 ml

RELATED PRODUCTS: • MitoShift™ Kit • TACS® Annexin V Kits • FlowTACS™ Kit • TACS® 2 FITC Kit

STORAGE: Store components at -20˚C and 4˚C.

REFERENCES: DePsipher™ 1. Moffitt, K.L., S.L. Martin, and B. Walker. 2010. From sentencing to execution – the processes of apoptosis. J Pharm Pharmacol. 62:547-562. 2. Cossarizza, A., M. Baccarani-Contri, G. Kalashnikova, and C. Franceschi. 1993. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenz-imidazol- carbocyanine iodide. Biochem. Biophys. Res. Commun. 197(1):40-45. 3. Gu, M., J.M. Cooper, J.W. Taanman, and A.H. Schapira. 1998. Mitochondrial DNA 141 transmission of the mitochondrial defect in Parkinson’s disease. Ann. Neurol. 44(2):177-186. 4. Mancini, M., M. Sedghinasab, K. Knowlton, A. Tam, D. Hockenbery, and B.O. Anderson. 1998. Flow cytometric measurement of mitochondrial mass and function: a novel method for assessing chemoresistance. Ann. Surg. Oncol. 5(3):287-295. 5. Salvioli, S., R. Maseroli, T.L. Pazienza, V. Bobyleva, and A. Cossarizza. 1998. Use of flow cytometry as a tool to study mitochondrial membrane potential in isolated, living hepatocytes. Biochemistry (Mosc.) 63:235-238.

PRODUCT CITATION: 1. Mungunsukh, O., A. J. Griffin, Y. H. Lee, and R. M. Day. 2010. Bleomycin Induces the Extrinsic Apoptotic Pathway in Pulmonary Endothelial Cells Am J Physiol Lung Cell Mol Physiol 298: L696-L703.

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APOPTOSIS DETECTION

Mitochondrial Events MitoShift™ Mitochondrial Potential Assay

In non-apoptotic, healthy cells, cellular energy produced during mitochondrial respiration is maintained as an electrochemical gradient that constitutes a high mitochondrial transmembrane ΔΨ potential ( mhigh). This membrane potential enables the cell to drive the synthesis of ATP and its disruption is associated with uncoupling of oxidative phosphorylation, generation of superoxide free radicals, and release of mitochondrial matrix-associated Ca2+ into the cytosol. Additionally, leakage of key apoptotic mitochondrial proteins such as cytochrome C, SMAC/Diablo and ΔΨ apoptosis inducing factor (AIF) have been associated with loss of m. Decreases in membrane potential have been used as a characteristic apoptotic marker.

There are several analyses compatible with tetramethylrhodamine ethyl ester (TMRE). Researchers ΔΨ use MitoShift™ (TMRE) to evaluate shifts in the m at the single mitochondrion level, by ΔΨ confocal microscopy. As m collapses, there is an outward flow of the dye along the altered pH gradient, leaving the mitochondria fluorescence free. WEHI 7.1 mouse lymphocytes at different cellular stages were stained with MitoShift™ and observed by fluorescence MitoShift™ can also be used in conventional fluorescence microscopy. The dye appears associated microscopy (Ex: 488 Em: 565nm). Healthy cells appear with with mitochondria in healthy cells, generally in the perinuclear area as red-orange, punctate punctate perinuclear fluorescence located in the fluorescence. In cells experiencing general mitochondrial depolarization, an efflux of the dye, from mitochondria; cells with depolarized mitochondria stain bright orange in the cytoplasm; necrotic or late apoptotic the mitochondria to the cytosol, results in brightly orange and diffused fluorescence. It has been cells do not emit any fluorescence. reported that the high dye concentration inside the mitochondria leads to a quenching of the fluorescence that is alleviated when the dye exits the organelle compartment. Early apoptotic cells

MitoShift™ will often be stained according to this pattern. A 142 Finally, in late apoptotic cells or cells that have lost their cellular membrane integrity, the dye is released into the medium and fluorescence is lost. This feature allows the use of MitoShift™ with flow cytometry to discriminate necrotic or late apoptotic cells from healthy cells.

FEATURES: • Simple. Just add MitoShift™ to media or reaction buffer. • Unique Stabilizer Solution included for sensitive cells. • Rapid. Takes a few minutes of hands-on time.

APPLICATIONS: B • Flow cytometry • Epifluorescence microscopy • Confocal microscopy

COMPONENTS: Catalog # Component Size 6305-100-01 MitoShift™* (1 mM) 100 µl 6305-100-02 Valinomycin 100 µl 6300-100-02 10X Reaction Buffer 30 ml 6300-100-03 Stabilizer Solution 5 ml

Healthy WEHI 7.1 mouse lymphocytes (A), and cells treated with etoposide for 2 hours with overnight recovery *Excitation/Emission (510–560/>590 nm) (B), were analyzed by flow cytometry using the PE channel. A distinct shift in the fluorescence occurs when the mitochondrial potential is disturbed in the late apoptotic cells.

ORDERING INFORMATION 6305-100-01 MitoShift™ (1 mM), 100 µl 6305-100-K MitoShift™ Mitochondrial Potential Assay, 100 Tests

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Mitochondrial Events MitoShift™ Mitochondrial Potential Assay

RELATED PRODUCTS: • DePsipher™ • TACS® Annexin V Kits • FlowTACS™ Kit

STORAGE: Store components at 4˚C.

REFERENCES: 1. Moffitt, K.L., S.L. Martin, and B. Walker. 2010. From sentencing to execution – the processes of apoptosis. J Pharm Pharmacol. 62:547-562. 2. Scaduto, R. and L.W. Grotyohann. 1999. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophysical Journal 76:469-477. 3. Fink, C., F. Morgan, and L.M. Loew. 1998. Intracellular fluorescent probe concentration by confocal microscopy. Biophysical Journal 75:1648-1658. 4. Ehrenberg, B., V. Montana, M.D. Wei, J. P. Wuskell, and L.M. Loew. 1988. Membrane MitoShift™ potential can be determined in individual cells from the Nernstian distribution of cationic dyes. Biophysical Journal 53:785-794.

PRODUCT CITATION: 1. Zhao, J., J. Chen, B. Lu, L. Dong, H. Wang, C. Bi, G. Wu, H. Guo, M. Wu, and Y. Guo. 2008. TIP30 Induces Apoptosis under Oxidative Stress through Stabilization of p53 Messenger RNA in Human Hepatocellular Carcinoma. Cancer Res 68:4133–4141. 143 2. Longo. M.,, F. Fiorito, G. Marfè, S. Montagnaro, G. Pisanelli, L. De Martino, G. Iovane and U. Pagnini. 2009. Analysis of apoptosis induced by Caprine Herpesvirus 1 in vitro. Virus Res. 145:227-235.

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Mitochondrial Events PARP/Apoptosis Assay Kits

During apoptosis, PARP-1 which catalyzes the NAD+-dependent addition of poly (ADP-ribose) (PAR) onto various cytoplasmic and nuclear proteins, is cleaved from about 116 kDa to 85 kDa. Moreover, this enzyme is a therapeutic target for BRCA1and BRCA2 associated breast cancers. The HT PARP/Apoptosis Assay is ideal for measuring the activity of PARP in cell extracts before and during apoptosis. This ELISA semi-quantitatively detects PAR deposited onto immobilized histone proteins in a 96-well format. An anti-PAR monoclonal antibody, goat anti-mouse IgG-HRP conjugate, and HRP substrate are used to generate a signal. Thus, signals correlate with PARP activity. Etoposide is a topoisomerase II inhibitor that stabilizes this enzyme after it Trevigen’s PARP Apoptosis Assay cleaves DNA. It is included as a control apoptosis inducer. Colorimetric or Chemiluminescent Detection FEATURES: • Colorimetric or Chemiluminescent readout HRP • 96 well format

Goat anti-mouse IgG-HRP • Highly sensitive – detects 0.1 mU PARP ~500 cells • Dynamic range between 0.1 to 10 mU PARP

Anti-PAR Monoclonal • Requires 10-100 ng extract for detection • Assay Time ~3 hrs

PAR APPLICATIONS: • Measure activity in peripheral blood leucocytes or tumor cells • Measure activity before and after apoptosis

PARP/APOPTOSIS ASSAY PARP/APOPTOSIS Histones • Measure effect of PARP inhibitors using cell extracts

144 KIT COMPONENTS: ASSAY DESIGN: PARP-HSA and Buffer PARP Cocktail Step 1: Ribosylation of histones by PARP. Activated DNA NAD+ • PAR polymer synthesized onto immobilized histones Histone-Coated Strip Wells PAR mAb and Diluent using PARP-HSA or cell extracts HRP Conjugate Etoposide • PARP inhibitors and caspase 3 cleavage prevent synthesis of PAR polymer. Colorimetric or Chemiluminescent Detection Reagents Step 2: Detection to measure incorporation of PAR attached to histones via PARP monoclonal antibody. REFERENCES: • Color/light output (Signal) is proportional to 1. Lawen A. 2003. Apoptosis—an introduction. BioEssays 25:888-896. PARP activity. 2. Okada H, Mak TW. 2004. Pathways of apoptotic and non-apoptotic death in tumor cells. • Monitor apoptosis by a decrease in PARP Signal. Nat Rev Cancer 4:592-603. 3. Miller MS, Zobre C, Lewis M. 1993. In vitro neuroprotective activity of inhibitors of poly-ADP ribose polymerase. Soc Neurosci Abstr 19.1656 4. Piper AA, Verma A, Zhang J, Snyder SH. 1999. Poly(ADP-ribose) polymerase, nitric oxide and cell death. Trends in Pharmacological Sciences 20:171-181. 5. Thiemermann C, Bowes J, Myint FP, and Vane JR. 1997. Inhibition of the activity of poly(ADP-ribose) synthase reduces ischemia-reperfusion injury in the heart and skeletal muscle. Proc Natl Acad Sci USA 94:679-683. 6. Virag L, Szabo C. 2002. The therapeutic potential of Poly(ADP-Ribose) Polymerase inhibitors. Pharmacological Reviews 54:375-429. 7. Baldwin EL, Osherhoff N. 2005. Etoposide, Topoisomerase II and Cancer. Curr. Med. Chem. Anti-Cancer Agents 5:363-372. 8. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, Kyle S, Meuth M, Curtin NJ, Helleday T. 2005. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434:913-7. 9. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, Santarosa M, ORDERING INFORMATION Dillon KJ, Hickson I, Knights C, Martin NM, Jackson SP, Smith GC, Ashworth A. 2005. 4684-096-K Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature HT Colorimetric PARP/Apoptosis 434:917-21. Assay Kit, 96 tests 10. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, Mortimer P, Swaisland H, Lau A, O'Connor MJ, Ashworth A, Carmichael J, Kaye SB, Schellens JH, de Bono JS. 2009. 4685-096-K HT Chemiluminescent PARP/Apoptosis Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. Assay Kit, 96 tests N Engl J Med 361:123-34.

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Cell Proliferation TACS® MTT Cell Proliferation Assay

The TACS® MTT Cell Proliferation Assay (MTT-CPA) is a sensitive kit for the measurement of cell proliferation based upon the reduction of the tetrazolium salt 3,[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT). Changes in cell proliferative activity caused by trophic factors, growth inhibitors, or inducers and inhibitors of apoptosis may be quantified using the MTT-CPA. MTT is reduced to an insoluble formazan dye by mitochondrial enzymes associated with metabolic activity. The reduction of MTT is primarily due to glycolytic activity within the cell and is dependent upon the presence of NADH and NADPH.

Common methods for determining cell viability depend upon membrane integrity (e.g. trypan blue exclusion), or incorporation of nucleotides during cell proliferation (e.g. BrdU or 3H-thymidine). These methods are limited by the impracticality of processing large numbers of samples, or by the requirement for handling hazardous materials. The MTT Assay, in contrast, provides a rapid and versatile method for assessing cell viability.

The assay is used to measure changes in cell proliferation. In actively proliferating cells, an increase in MTT conversion is spectrophotometrically quantified. Comparison of this value to an untreated control provides a relative increase in cellular proliferative activity. Conversely, in cells MTT ASSAY that are undergoing apoptosis, MTT reduction decreases, reflecting the loss of cell viability.

FEATURES: • Convenient. Stabilized formulation is stored in your refrigerator and does not require thawing before use. • Non-isotopic. Assay for cell proliferation, cytotoxicity, and viability does not require isotopic reagents. 145 • Fast. High throughput microplate format. • Flexible. The reaction product can be visualized directly by microscopy to evaluate cell to cell reactivity, or solubilized and evaluated by microplate reading. • Safe. Reaction product is solubilized using a non-organic solvent.

APPLICATIONS: • Cell proliferation assays • Cytotoxicity analysis • Apoptosis screening

COMPONENTS: Catalog # Component Size 4890-25-01 MTT Reagent 25 ml 4890-25-02 Detergent Reagent 250 ml

ITEMS NOT INCLUDED: Microwell plate

RELATED PRODUCTS: TACS® XTT Cell Proliferation Assay Calcein AM Cell Viability Kit

ORDERING INFORMATION STORAGE: Components are stored at 4˚C and room temperature. 4890-25-01 MTT Reagent, 25 ml REFERENCES: 4890-25-02 1. Alley, M.C., D.A. Scudiero, A. Monks, M.L. Hursey, M.J. Czerwinsky, D.L. Fine, B.J. Abbott, Detergent Reagent, 250 ml J.G. Mayo, R.H. Shoemaker, and M.R. Boyd. 1988. Feasibility of drug screening with panels 4890-25-K of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 48:589-601. MTT Cell Proliferation Assay, 2500 Wells 2. Mosman, T. 1983. Rapid colorimetric assay for cellular growth and survival: application to 4890-50-K proliferation and cytotoxicity assays. J. Immunol. Methods 65:55-63. MTT Cell Proliferation Assay, 5000 Wells 3. van de Loosdrecht, A.A., R.H.J. Beelen, G.J. Ossenkoppele, M.G. Broekhoven, and M.M.A.C. Langenhuijsen. 1994. A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid 1-800-873-8443 • www.trevigen.com leukemia. J. Immunol. Methods 174:311-320. INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:22 PM Page 150

APOPTOSIS DETECTION

Cell Proliferation TACS® XTT Cell Proliferation Assay

The use of tetrazolium salts, including XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H- tetrazolium-5-carboxanilide), to assay cell proliferation, cell viability, and/or cytotoxicity is a widespread, established practice. The procedures avoid radioactivity, allow for rapid determination in microplates, and give reproducible and sensitive results. Cleavage of the tetrazolium salt to formazan occurs via the succinate-tetrazolium reductase system in the mitochondria of metabolically active cells. The reaction is attributed mainly to mitochondrial enzymes and electron carriers, but a number of other non-mitochondrial enzymes have been implicated.

XTT, a yellow tetrazolium salt, is cleaved to a soluble orange formazan dye, which can be measured by absorbance at 490 (or 450) nm in a microplate reader. Efficient reduction of XTT requires an electron coupling reagent. This kit includes both XTT and the electron coupling reagent for a convenient and simple assay.

FEATURES: • Sensitive • No radioactivity • Rapid (no solubilization step as in an MTT assay) • Ideal for high throughput assays (no washing or other steps that can cause cell loss and variability)

APPLICATIONS: XTT ASSAY • Cell proliferation assays • Cytotoxicity analysis 146 • Apoptosis screening

COMPONENTS: Catalog # Component Size 4891-025-01 XTT Reagent 5 x 25 ml 4891-025-02 XTT Activator 5 x 0.5 ml

RELATED PRODUCTS: TACS® MTT Cell Proliferation Assay Calcein AM Cell Viability Kit

STORAGE: Components are stored at -20˚C.

REFERENCES: 1. Cole, S.P.C. 1986. Cancer Chemother. Pharmacol. 17:259-263. 2. Twentyman, P.R., et al. 1987. Br. J. Cancer 56:279-285. 3. Scudiero, D., et al. 1988. Cancer Res. 48:4827-4833. 4. Behl, C., et al. 1994. Cell 77:817-827. 5. Roehm, N. et al. 1991. J. Immunol. Meth. 142:257-265. 6. Weislow, O., et al. 1989. J. Natl. Cancer Inst. 81:577-586.

Quantitation of HT-1080 cells using XTT. HT-1080 cells were serially diluted in DMEM and incubated for ORDERING INFORMATION two hours with XTT. Absorbance values were obtained at 490 nm in 4891-025-01 a 96 well plate reader. XTT Reagent, 5 X 25 ml 4891-025-02 XTT Activator, 5 X 0.5 ml 4891-025-K XTT Cell Proliferation Assay Kit, 2500 Wells

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Cell Proliferation Calcein AM Cell Viability Kit

The Calcein AM Kit provides a simple, rapid, and accurate method to measure cell viability and/or cytotoxicity. Calcein AM is a non-fluorescent, hydrophilic compound that easily permeates intact, live cells. The hydrolysis of Calcein AM by intracellular esterases produces calcein, a hydrophilic, strongly fluorescent compound that is well retained in the cell cytoplasm. Cells grown in black-walled plates can be stained and quantified in less than two hours.

FEATURES: • Suitable for proliferating and non-proliferating cells. • Ideal for both suspension and adherent cells. • Non-radioactive microplate. • Rapid (no solubilization step as in an MTT assay). Figure 1. Calcein AM Quantification of Jurkat Cells. Jurkat • Ideal for high throughput assays. cells were grown in RPMI supplemented with 10% FBS, • Better retention and brightness compared to other fluorescent compounds (i.e. fluorescein). washed with 1X Calcein AM DW Buffer, and counted using • Useful in a variety of studies, including: cell adhesion, chemotaxis, multi-drug resistance, Trypan blue and a hemacytometer. Cells were serially cell viability, apoptosis, and cytotoxicity. diluted in a black-walled microplate and then incubated with 1 µm Calcein AM for 30 minutes at 37˚C under 5% • Adaptable to a wide variety of techniques, including: microplate assays, CALCEIN AM CO2. Fluorescence values were obtained using a 484 nm immunocytochemistry, flow cytometry, and in vivo cell tracing. excitation filter and a 520 nm emission filter in a BMG Laboratories’ FluoStar Optima Fluorometer with a gain APPLICATIONS: setting of 1600. • Cytotoxicity analysis • Cell viability assay • Cell staining

COMPONENTS: 147 Calcein AM Kit.

Catalog # Component Size 4892-010-01 Calcein AM 2 x 50 µg 4892-010-02 10X Calcein AM DW Buffer 200 ml

RELATED PRODUCTS: • MTT Cell Proliferation Assay • XTT Cell Proliferation Assay

Calcein AM STORAGE: The fluorescent dyes used for quantitatively determining Components are stored at 4˚C and -20˚C. viable cells (Calcein AM) may also be used for analyzing structural formation using fluorescent microscopy. REFERENCES: The figure above reveals formation of acinar structures by 1. Wang, X.M., et al. 1993. H. Immunol. 37(4):264-270. PC-3 prostatic adenocarcinoma cells grown in vitro on 3-D 2. Bharti, A.C., et al. 2004. J. Biol. Chem. 279(7):6065-6076. Culture Matrix™ Basement Membrane Extract (see page 6) using: (A) Calcein AM,. Trevigen has also developed a 3. Poncet, D., et al. 2003. Apoptosis 8(5):521-530. colorimetric assay for cell staining (see Culturex® Cell 4. Bell, E., et al. 2003. Diabetes 52(11):2731-2739. Staining Kit, page 104). 5. Weston, S.A., et al. 1990. J. Immunol. Methods 133:87-97. 6. Yang, A., et al. 2002. Cell Biol. Toxicol. 18(2):97-108. 7. Eneroth, A., et al. 2001. Eur. J. Pharm. Sci. 12(3):205-214. 8. Braut-Boucher, F., et al. 1995. J. Immunol. Methods 178(1):41-51.

ORDERING INFORMATION 4892-010-01 Calcein AM, 2 X 50 µg 4892-010-02 10X Calcein AM DW Buffer, 200ml 4892-010-K Calcein AM Cell Viability Assay Kit, 1000 Wells

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Apoptosis Antibody Selection Guide

Applications† Catalog # Description Clone Size Specificity Subtype IHC WB IP Page

Apoptosis 2280-MC-100 Anti-Bax YTH-5B7 100 µg M, IgG1 • * • • 149 2281-MC-100 Anti-Bax YTH-6A7 100 µg H, M, R IgG1 • * • • 149 2282-MC-100 Anti-Bax YTH-2D2 100 µg H IgG1 • * • • 149 2290-MC-100 Anti-Bcl-2 YTH-10C4 100 µg M, R IgG1 • • 151 2291-MC-100 Anti-Bcl-2 YTH-8C8 100 µg H IgG1 • • 151 2300-MC-100 Anti-Bcl-XL YTH-2H12 100 µg H, M, R IgG2a • • • 152 2305-PC-020 Anti-Cleaved Caspase-3 Polyclonal (AP) 20 µl H, M n/a • • • 154 2305-PC-100 Anti-Cleaved Caspase-3 Polyclonal (AP) 100 µl H, M n/a • • • 154 6361-PC-100** Anti-H/M PBR Polyclonal 100 µl H, M, R n/a • • 153 6362-PC-100** Anti-M PBR Polyclonal 100 µl M n/a • • 153

Control 2275-PC-100 Anti-G3PDH Polyclonal 100 µl H, M n/a • • 155 2275-PC-020 Anti-G3PDH Polyclonal 20 µl H, M n/a 155

APOPTOSIS ANTIBODY SELECTION GUIDE

148 AP = Affinity Purified IHC = Immunohistochemistry; WB = Western Blot; IP = Immunoprecipitation; Mm = Mammalian; H = Human; M = Mouse; R = Rat; Y = Yeast; D = D. melanogaster; X = X. laevis; n/a = not applicable; † Data may not yet be available for each antibody for all applications for all antibodies. * In a cell system that overexpressed BAX protein ** Control protein available

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Antibodies Anti-Bax Monoclonal Antibodies

Bax, a 21 kDa eukaryotic protein, plays an important role in the regulation of cell death in a number of eukaryotic cells. The over-expression of Bax has been shown to accelerate cell death. The ratio of Bax to other Bcl-2 family members and its subcellular distribution is thought to help regulate the process of programmed cell death.

Anti-Bax (clone YTH-5B7) Catalog # 2280-MC-100

IMMUNOGEN: The antibody was raised against a synthetic peptide corresponding to amino acids 3 to 16 of the mouse Bax sequence.

SPECIFICITY: 5B7: Western blot analysis of mouse Wehi (W) and human The YTH-5B7 clone recognizes both native and detergent-treated mouse Bax protein. HT1080 (H) cell lines. Cells were lysed in Tris-Glycine SDS 7 sample buffer at a concentration of 1 x 10 cells/ml, and 10 SUBTYPE: µl of lysates were loaded per well of a 4-20% Tris-Glycine

gel. Proteins were transferred onto an Immobilon FL IgG1 ANTI-BAX membrane, detected using Trevigen’s anti-Bax (YTH-5B7) antibody (cat# 2280-MC-100) and visualized using an IR800- Anti-Bax (clone YTH-6A7) conjugated secondary antibody. The membrane was scanned Catalog # using an Odyssey Infrared Imaging System (Licor). Trevigen’s Bax antibody (clone YTH-5B7) (cat# 2280-MC- 2281-MC-100 100) recognizes mouse, but not human Bax protein. IMMUNOGEN: The antibody was raised against a synthetic peptide corresponding to amino acids 12 to 24 149 of the human Bax sequence.

SPECIFICITY: The YTH-6A7 clone is conformation dependent, and will recognize only detergent-treated human, mouse, or rat Bax protein and not the native form.

SUBTYPE: IgG1

Anti-Bax (clone YTH-2D2) Catalog # 2282-MC-100

IMMUNOGEN: The antibody was raised against a synthetic peptide corresponding to amino acids 3 to 16 of the human Bax sequence.

6A7: Western blot analysis of mouse Wehi (W) and human SPECIFICITY: HT1080 (H) cell lines. Cells were lysed in Tris-Glycine SDS sample buffer a concentration of 1 x 107 cells/ml and 10 µl The YTH-2D2 clone will recognize both native and detergent-treated human Bax protein. of lysates were loaded per well of a 4-20% Tris-Glycine gel. Proteins were transferred onto an Immobilon FL membrane, SUBTYPE: and mouse and human Bax proteins were detected using IgG Trevigen’s anti-Bax (YTH-6A7) antibody (catalog # 2281-MC- 1 100). Bands were visualized using an IR800-conjugated secondary antibody. The membrane was scanned using an PREPARATION: Odyssey Infrared Imaging System (Licor). The Bax antibodies are purified from mouse ascites and are provided as 100 µg aliquots in PBS buffer containing 0.01% sodium azide.

APPLICATIONS: • Western blotting (dilution factor 1:1,000) • Immunoprecipitation • Immunohistochemistry*

*In cell systems that over-express Bax.

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Antibodies Anti-Bax Monoclonal Antibodies

RELATED PRODUCTS: Section on Bcl-2 family antibodies (see page 151).

STORAGE: Store at 4˚C.

REFERENCES: 1. Hsu, Y.-T., K.G. Wolter, and R.J. Youle. 1997. Cytosol-to-membrane redistribution of Bax and Bcl-XL during apoptosis. Proc. Natl. Acad. Sci. 94:3668-3672. 2. Hsu, Y.-T. and R.J. Youle. 1997. Nonionic detergents induce dimerization among members 2D2: Western blot analysis of mouse Wehi (W) and human of the Bcl-2 Family. J. Biol. Chem. 272:13829-13834. HT1080 (H) cell lines. Cells were lysed in Tris-Glycine SDS sample buffer at a concentration of 1 x 107 cells/ml and 10 3. Neuchushtan, A., C.L. Smith, Y.-T. Hsu, and R.J. Youle. 1999. Conformation of the Bax µl of lysates were loaded per well of 4-20% Tris-Glycine gel. C-terminus regulates subcellular location and cell death. EMBO. J. 18:2330-2341. Proteins were transferred onto an Immobilon FL membrane and Bax was detected with Trevigen’s anti-Bax (YTH-2D2) PRODUCT CITATIONS: antibody (cat# 2282-MC-100) and visualized using an IR800- conjugated secondary antibody. The membrane was scanned 1. Wakabayashi, Y., H. Watanabe, J. Inoue, N. Takeda, J. Sakata, Y. Mishima, J. Hitomi, T. using an Odyssey Infrared Imaging System (Licor). Yamamoto, M. Utsuyama, O. Niwa, S. Aizawa, and R. Kominami. 2003. Bcl11b is required for Trevigen’s Bax antibody (clone YTH-2D2) (catalog # 2282- differentiation and survival of αβT lymphocytes. Nat. Immunol. 4:533-539. MC-100) recognizes human, but not mouse Bax protein. 2. Kurosu, T., M. Ohki, N. Wu, H. Kagechika, and O. Miura. 2009. Sorafenib induces apoptosis specifically in cells expressing BCR/ABL by inhibiting its kinase activity to activate the intrinsic

ANTI-BAX mitochondrial pathway. Cancer Res. 69:3927-3936. 3. Gillissen, B., J. Wendt, A. Richter, A. Richter, A. Müer, T. Overkamp, N. Gebhardt, R. Preissner, 150 C. Belka, B. Dörken, and P.T. Daniel. 2010. Endogenous Bak inhibitors Mcl-1 and Bcl-xL: differential impact on TRAIL resistance in Bax-deficient carcinoma J. Cell Biol. 188:851–862.

ORDERING INFORMATION

2280-MC-100 Anti-Mouse Bax Monoclonal Antibody (clone YTH-5B7), 100 µg 2281-MC-100 Anti-Bax Monoclonal Antibody (clone YTH-6A7), 100 µg 2282-MC-100 Anti-Human Bax Monoclonal Antibody (clone YTH-2D2), 100µg

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Antibodies Anti-Bcl-2 Monoclonal Antibodies

The Bcl-2 family of proteins plays a crucial role in the regulation of cell death in many eukaryotic systems. The over-expression of Bcl-2, a 26 kDa protein, has been shown to promote cell survival and the ratio of Bcl-2 to other Bcl-2 family members is believed to modulate the apoptotic process.

Anti-Bcl-2 (clone YTH-10C4) Catalog # 2290-MC-100

IMMUNOGEN: The antibody was raised against a synthetic peptide corresponding to amino acids 61 to 76 of the mouse Bcl-2 sequence.

10C4: Western blot analysis of L929 and Jurkat (J) cell lines. SPECIFICITY: Cells were lysed in Tris-Glycine SDS sample buffer at the concentration 107 cells/ml and 10 µl of lysates were loaded The YTH-10C4 clone recognizes mouse and rat Bcl-2 protein.

per well of a 4-20% Tris-Glycine gel. Proteins were ANTI-BCL-2 transferred onto an Immobilon FL membrane and Bcl-2 SUBTYPE: protein was detected with Trevigen’s anti-Bcl-2 (YTH-10C4) IgG anti-body (catalog # 2290-MC-100) followed by an IR800- 1 conjugated secondary antibody. The membrane was scanned using an Odyssey Infrared Imaging System (Licor). Anti-Bcl-2 (clone YTH-8C8) Catalog # 2291-MC-100

IMMUNOGEN: 151 The antibody was raised against a synthetic peptide corresponding to amino acids 41 to 54 of the human Bcl-2 sequence.

SPECIFICITY: The YTH-8C8 clone recognizes human Bcl-2 protein.

SUBTYPE: IgG1 RELATED PRODUCTS: Anti-Bax Monoclonal Antibodies (see page 150); Anti-Bcl-XL (see page 152)

STORAGE: 8C8: Western blot analysis of Wehi (W) and Jurkat (J) cell Store at 4˚C. lines. Cells were lysed in Tris-Glycine SDS sample buffer at a concentration of 1 x 107 cells/ml and 10 µl of lysates were REFERENCES: loaded per well of 4-20% Tris-Glycine gel. Proteins were 1. Hsu, Y.-T., K.G. Wolter, and R.J. Youle. 1997. Cytosol-to-membrane redistribution of Bax and transferred onto an Immobilon FL membrane and Bcl-2 protein was detected with Trevigen’s anti-Bcl-2 (clone YTH- Bcl-XL during apoptosis. Proc. Natl. Acad. Sci. 94:3668-3672. 8C8) antibody (catalog # 2291-MC-100) and visualized using 2. Hsu, Y.-T. and R.J. Youle. 1997. Nonionic detergents induce dimerization among members of an IR800-conjugated secondary antibody. The membrane the Bcl-2 Family. J. Biol. Chem. 272:13829-13834. was scanned using an Odyssey Infrared Imaging System 3. Wolter, K.G., Y.-T. Hsu, C.L. Smith, A. Nechushtan, X.-G. Xi, and R.J. Youle. 1997. Movement (Licor). Trevigen’s Bcl-2 antibody (clone YTH-8C8) of Bax from the cytosol to the mitochondria during apoptosis. J. Cell. Biol. 139:1281-1292. (catalog # 2291-MC-100) recognizes human Bcl-2 protein.

PRODUCT CITATION: 1. Mena, S., M. Benlloch, A. Ortega, J. Carretero, E. Obrador, M. Asensi, I. Petschen, B.D. Brown, and J.M. Estrela. 2007. Bcl-2 and glutathione depletion sensitizes B16 melanoma to combination ORDERING INFORMATION therapy and eliminates metastatic disease. Clin Cancer Res. 13:2658-2666. 2290-MC-100 Anti-Mouse Bcl-2 Monoclonal Antibody (clone YTH-10C4), 100 µg 2291-MC-100 Anti-Human Bcl-2 Monoclonal Antibody (clone YTH-8C8), 100 µg

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Antibodies Anti-Bcl-XL Monoclonal Antibody

The Bcl-2 family of proteins plays a crucial role in the regulation of cell death in many eukaryotic systems. Bcl-XL is a 28 kDa protein associated with cell survival. Bcl-X is reported in two forms, Bcl-XL and Bcl-XS. The short form is reported to inhibit the survival promoting activity of the long form. Bcl-XS is a splice variant at the RNA level, but its functional role is not clear.

Anti-Bcl-XL (YTH-2H12) Catalog # 2300-MC-100

IMMUNOGEN: The antibody was raised against a synthetic peptide corresponding to amino acids 3 to 14 of the human Bcl-XL sequence.

SPECIFICITY: The YTH-2H12 clone recognizes human, mouse, and rat Bcl-XL protein. Figure Legend: Western blot analysis of mouse Wehi (W) and human Jurkat (J) cell lines. Cells were lysed in SUBTYPE: Tris-Glycine SDS sample buffer at 107 cells/ml and 10 µl of lysates were loaded per well of a 4-20% Tris-Glycine gel. IgG2a Proteins were transferred onto an Immobilon FL membrane and mouse and human Bcl-XL proteins were PREPARATION: detected with anti-Bcl-XL (YTH-2H12) antibody (catalog # The Bcl-XL antibody is purified from mouse ascites and is provided as a 100 µg aliquot in 2300- MC-100) followed by IR800-conjugated secondary PBS buffer containing 0.01% sodium azide. antibody. The membrane was scanned using an Odyssey ANTI-BCL-XL Infrared Imaging System (Licor). APPLICATIONS: 152 • Western blotting (dilution factor 1:1,000) • Immunohistochemistry • Immunoprecipitation

STORAGE: Store at 4˚C.

RELATED PRODUCTS: Anti-Bax Monoclonal Antibodies (see page 149)

REFERENCES: 1. Hsu, Y.-T., K.G. Wolter, and R.J. Youle. 1997. Cytosol-to-membrane redistribution of Bax and Bcl-XL during apoptosis. Proc. Natl. Acad. Sci. USA 94:3668-3672. 2. Hsu, Y.-T. and R.J. Youle. 1997. Nonionic detergents induce dimerization among members of the Bcl-2 Family. J. Biol. Chem. 272:13829-13834. 3. Wolter, K.G., Y.-T. Hsu, C.L. Smith, A. Nechushtan, X.-G. Xi, and R.J. Youle. 1997. Movement of Bax from the cytosol to the mitochondria during apoptosis. J. Cell. Biol. 139:1281-1292. 4. Chu, W.-S., N.S.I. Aguilera, M.Q. Wei, S.L. Abbondanzo. 1999. Antiapoptotic marker, Bcl-XL, expression on Reed-Sternberg cells of Hodgkin’s disease using a novel monoclonal marker, YTH-2H12. Hum. Pathol. 30:1065-1070.

PRODUCT CITATION: 1. Sarosiek, K.A., R. Malumbres, H. Nechushtan, A.J. Gentles, E. Avisar, and I.S. Lossos. 2010. Novel IL-21 signaling pathway up-regulates c-Myc and induces apoptosis of diffuse large B-cell lymphomas. Blood. 115:570-580.

ORDERING INFORMATION

2300-MC-100 Anti-Bcl-XL Monoclonal Antibody (clone YTH-2H12), 100 µg

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Antibodies Anti-PBR Polyclonal Antibodies

The peripheral-type benzodiazepine receptor (PBR) is a ubiquitous 18 KDa protein involved in the A regulation of cholesterol transport from the outer to inner mitochondrial membrane. Thought to play a role in the formation of the mitochondrial permeability transition (PT) pore involved in apoptotic mechanisms, PBR is also present in abundance in tumors, in aggressive breast cancer cell lines, metastatic human breast tumor biopsy sections, astrocytomas and other brain tumors. PBR over-expression contributes to hormone-induced tumor cell proliferation, an important contributor to morbidity in cancer.1

IMMUNOGEN: The anti-PBR antibody was developed in rabbits against an internal sequence of the PBR protein

consisting of amino acids 71 to 88 of the human PBR sequence. The specificity of the antibody ANTI-PBR AND CONTROL was assessed in steroidogenic cells and in mitochondrial extracts.

SOURCE: Rabbit

SPECIFICITY: Catalog # 6361-PC-100 recognizes mouse, rat, and human PBR protein. Catalog # 6362-PC-100 recognizes mouse PBR protein.

PREPARATION: PBR antibody is provided as a pooled rabbit anti-sera in 1X PBS. Legend: Western Blot analysis of PBR where 1 µg of the recombinant PBR protein was loaded on a 12% SDS-PAGE gel, transferred to a PVDF membrane and detected with a APPLICATIONS: 1:1000 dilution of the anti-PBR anti-body (catalog # 6362- • Immunohistochemistry 153 PC-100). Detection was performed with a peroxidase- • Western blotting (dilution factor 1:1,000) conjugated secondary antibody to rabbit followed by chemiluminescence. STORAGE: -20°C PBR Protein (Recombinant)

CONTROL: Recombinant Peripheral benzodiazepine receptor (PBR) protein.

SOURCE: Recombinant full length mouse peripheral benzodiazepine receptor (PBR) purified from E. coli.

PREPARATION: Purified protein is provided at 0.2 mg/ml in: 20 mM Tris 7.8, 1 mM EDTA, 0.1% Sodium Lauryl Sarcosine.

EXPECTED SIZE: The recombinant protein is a fusion protein with a molecular weight of 20.4 kDa. Please note that multimers of the proteins are commonly seen on gels and Western Blotting Detection. The native protein is 18 kDa. (Figure A).

REFERENCES: ORDERING INFORMATION 1. Li W, Hardwick MJ, Rosenthal D, Culty M, Papadopoulos V. 2007. Peripheral-type benzodiazepine receptor overepression and knockdown in human breast cancer cells 6361-PC-100 indicate its prominent role in tumor cell proliferation. Biochem Pharmacol 73:491-503. Anti-Human/Mouse PBR Antibody, 100 µl 2. Amri H, Ogwuebu SO, Boujrad N, Drieu K, Papadopoulos V. 1996. In vivo regulation of 6362-PC-100 peripheral-type benzodiazepine receptor and glucocorticoid synthesis by Ginkgo biloba Anti- Mouse PBR Antibody, 100 µl extract EGb 761 and isolated ginkgolides. Endocrinology 137: 5707–5718.

6360-025-01 PRODUCT CITATION: PBR Protein (Recombinant), 25 µl 1. Costa, B., A. Salvetti, L. Rossi, F. Spinetti, A. Lena, B. Chelli, M. Rechichi, E. Da Pozzo, V. Gremigni, and C. Martini. 2006. Peripheral benzodiazepine receptor: characterization in 1-800-873-8443 • www.trevigen.com human T-lymphoma Jurkat cells. Mol Pharmacol. 69:37-44. INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:22 PM Page 158

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Antibodies Anti-Cleaved Caspase-3 Rabbit Polyclonal Antibody

The caspases are a family of structurally-related cysteine proteases that are synthesized as inactive precursors that have to be cleaved at key aspartate residues to be activated. These enzymes function 30 kDa in a proteolytic cascade in which caspases with a long prodomain act upstream of those with a short prodomain (procaspase 3, 6, and 7). In Fas-mediated apoptosis, caspase 3 plays an important role downstream of caspase 8.

18 kDa Following induction of apoptosis, proteolytic cleavage of procaspase 3 occurs to generate an active 18 kDa caspase 3 fragment, which targets key modulators of the apoptotic pathway, including poly-ADP-ribose polymerase and other caspases, for cleavage. Trevigen’s antibody recognizes the active 18 kDa fragment but does not recognize the 32 kDa procaspase 3, and is therefore useful as 0 3h 5h a specific marker for apoptosis.

Immunoblot of SDS-extracts from mouse cells treated with 25 µM IMMUNOGEN: etoposide. Samples were electrophoresed on a 12%Tris-Glycine gel and blotted onto a PVDF membrane. The 18 kDa cleaved This antibody is a rabbit polyclonal generated by immunization with a 13 amino acid peptide caspase 3 was detected by Trevigen’s anti-cleaved caspase 3 sequence from carboxyl terminus of the human and mouse caspase 3 p18 subunit. followed by anti-rabbit conjugated to horseradish peroxidase and chemiluminescence. The 30 kDa band is not the procaspase 3. SPECIFICITY: The antibody detects human and mouse cleaved caspase 3.

PREPARATION: This antibody is provided as affinity purified IgG fraction in phosphate buffered saline with ANTI-CLEAVED CASPASE-3 ANTI-CLEAVED 0.01% sodium azide.

154 APPLICATIONS: • Immunocytochemistry/Immunohistochemistry • Western blotting (dilution factor 1:1,000) • Immunoprecipitation

RELATED PRODUCTS: TACS® Laddering Kit EtBr

STORAGE: Can be stored for 1 month at 4°C. Freeze in working aliquots at -20˚C in a manual defrost freezer to avoid repeated freeze-thawings.

Jurkat cells were treated with 25 µM etoposide for 5 hours to REFERENCE: induce apoptosis. Cells were labeled with 1:250 dilution of 1. Srinivasan, A., K.A. Roth, R.O. Sayers, K.S. Shindler, A.M. Wong, L.C. Fritz, and K.J. Anti-Cleaved Caspase 3 Rabbit Polyclonal Antibody followed by Tomaselli. 1998. In situ immunodetection of activated caspase-3 in apoptotic neurons in the anti-rabbit horseradish peroxidase. Detection performed with developing nervous system. Cell Death Diff. 5:1004-1016. Trevigen’s TACS™ Blue Label and counterstained with Nuclear Fast Red. PRODUCT CITATIONS: 1. Henneke I., S. Greschus, R. Savai, M. Korfei, P. Markart, P. Mahavadi, R.T. Schermuly, M. Wygrecka, J. Stürzebecher, W. Seeger, A. Günther, and C. Ruppert. 2010. Inhibition of urokinase activity reduces primary tumor growth and metastasis formation in a murine lung carcinoma model. Am J Respir Crit Care Med. 181:611-619. 2. Miura T, Chiba M, Kasai K, Nozaka H, Nakamura T, Shoji T, Kanda T, Ohtake Y, Sato T. 2008. Apple procyanidins induce tumor cell apoptosis through mitochondrial pathway activation of ORDERING INFORMATION caspase-3. Carcinogenesis. 29:585-593. 2305-PC-020 Anti-Cleaved Caspase 3 Rabbit Polyclonal Antibody, 20 µl 2305-PC-100 Anti-Cleaved Caspase 3 Rabbit Polyclonal Antibody, 100 µl

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Antibodies Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Rabbit Polyclonal Antibody

CATALOG #: 2275-PC-100

DESCRIPTION: The abundance of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) in eukaryotic cells is relatively unaffected by external factors. In Western blot analysis the level of G3PDH, a ~38 kDa protein, can be used as a reference value for comparisons between different cell lysates or gel loadings.

PHYSICAL STATE: Purified IgG is provided in phosphate buffered saline without preservative.

IMMUNOGEN: A synthetic peptide corresponding to a portion of the human G3PDH sequence. ANTI-G3PDH IG CLASS: G3PDH-specific rabbit IgG.

SPECIFICITY: Cell lines vary regarding their G3PDH content. Cell lysates The antibody detects human and mouse G3PDH (other species not tested). from L292 cells (lane 1), WEHI 7.1 cells (lane 2), and INT 407 cells (lane 3) were analyzed by SDS-PAGE and Western STORAGE CONDITIONS: blot using a 1:1000 dilution of G3PDH in 5% nonfat milk in 155 PBS, 0.05% Tween®20. The blot was developed using HRP This antibody can be stored at -20°C or -80°C. Avoid repeated freeze-thawing by aliquoting into membrane solution. smaller portions.

APPLICATIONS: For Western blotting and immunoprecipitation, an antibody dilution of between 1:1000 and 1:5000 is recommended. Empirical testing may be required.

REFERENCES: 1. Arcari P, Martinelli R, Salvatore F. (1984) The complete sequence of a full length cDNA for human liver glyceraldehydes-3-phosphate Dehydrogenase: evidence for multiple mRNA species. Nucleic Acids Research 12:9179-9189.

ORDERING INFORMATION

2275-PC-020 Anti-h/m G3PDH Antibody, 20 µl 2275-PC-100 Anti-h/m G3PDH Antibody, 100 µl

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Apoptosis Research Accessories

Apoptosis Inducers Valinomycin, Etoposide, Staurosporine

All three are known to induce apoptosis. Valinomycin is used to induce mitochondrial potential (Δψm) disruption. Etoposide is a DNA synthesis inhibitor that induces double-stranded and ORDERING INFORMATION single-stranded DNA breaks. Staurosporine is a phospholipid/calcium-dependent protein kinase 6305-100-02 inhibitor that prevents ATP binding. Valinomycin, 100 µl Apoptosis Grade™ PBS Buffer and Water 4886-400-01 Etoposide, 400 µl The 10X PBS buffer is a high quality preparation manufactured without magnesium or cal- 4886-100-02 cium, and is suitable for cell resuspension and washing in the in situ apoptosis detection Staurosporine, 100 µl (TUNEL) procedure, CometAssay® (Cat# 4250-50-K) applications, and a variety of other applications requiring ultrapure PBS buffer. 4870-500 Apoptosis Grade™ 10X PBS, pH 7.4, The water is purified through a reverse osmosis system and multistage nanoparticle filtration 500 ml system assuring greater than 18 megohm-cm2 resistivity is provided in a convenient 500 ml 4870-500-6 size. The water is then sterilized using a unique autoclaving process to assure the highest Apoptosis Grade™ 10X PBS, pH 7.4, quality. 6 x 500 ml Controls 4869-500 Apoptosis Grade™ Water, 500 ml Cell Culture Control Slides, Tissue Control Slides, EpiDerm™ Control Slides, TACS Nuclease™ and Buffer

APOPTOSIS RESEARCH ACCESSORIES 4869-500-6 Apoptosis Grade™ Water, 6 x 500 ml Trevigen offers several different types of control slides to develop familiarity with the methods 156 4800-30-20 of in situ apoptosis labeling and to determine if reagents are working optimally. Cell Culture Cell Culture Control Slides, 2 slides Control Slides are useful for researchers studying apoptosis in cell culture systems, whereas 4800-30-21 the Tissue Control Slides are helpful when investigating tissue specimens. EpiDerm™ Control Cell Culture Control Slides, 5 slides Slides (Cat# 4800-30-42) are sections of synthetic human skin tailored for use with DermaTACS™. 4800-30-40 Tissue Control Slides, 2 slides TACS-Nuclease™ is a novel DNA endonuclease that is used to prepare a positive control from 4800-30-41 your own samples. The nuclease can simply be added to the labeling reaction mixture or if Tissue Control Slides, 5 slides preferred in a separate step. DNA fragments formed by the action of this enzyme serve as 4800-30-42 substrates for labeling, producing a positive signal in virtually every cell. EpiDerm™ Control Slides, 2 slides 4800-30-N Counterstains TACS Nuclease™,15 µl, and Buffer Blue Counterstain, Nuclear Fast Red, Methyl Green, Red Counterstain C

4820-30-13 Trevigen offers a variety of counterstains for use in our TACS® assay kits. Blue Counterstain, Blue Counterstain, 50 ml unlike Methyl Green, works well in neuronal tissue, and is also a good choice for double- 4800-30-17 labeling with brown or red detection systems. Nuclear Fast Red is the ideal counterstain for Nuclear Fast Red, 50 ml Trevigen kits employing the TACS Blue Label™ chromogenic substrate. Red Counterstain C 4800-30-18 provides an alternative for a red counterstain. Our Methyl Green stain is free of crystal violet Methyl Green, 50 ml contamination and provides excellent contrast with DAB detection systems. 4800-30-19 Red Counterstain C, 50 ml Mounting Media Mounting Medium 4862-10 Glass Coverslips 22 x 60 mm, Trevigen Mounting Medium is a high quality, optically-clear, toluene-based mounting 500 each medium-- ideal for mounting samples processed with our TACS® In Situ Apoptosis Detection 4865-25 kits using either DAB or TACS Blue Label™. Mounting samples protects the specimen from Mounting Medium, 25 ml damage and produces a clear image for microscopic visualization and photography. 4866-20 For fluorescent samples, use Trevigen Fluorescence Mounting Medium (Cat# 4866-20). Fluorescence Mounting Medium, 20 ml Fluorescence Mounting Medium 4867-100 ® Hydrophobic Coverslips 22 x 40 mm, Trevigen Fluorescence Mounting Medium is specially designed for use with our TACS In Situ 100 each Apoptosis Detection kits that use fluorescein as the detection method to minimize photobleaching that can occur during visualization of the samples.

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APOPTOSIS DETECTION

Apoptosis Research Accessories

Permeabilization Reagents for In Situ Apoptosis Detection Proteinase K

Proteinase K is used for permeabilizing cells and tissues prior to labeling using any of the TACS® In Situ Apoptosis Detection kits.

NeuroPore™ APOPTOSIS RESEARCH ACCESSORIES

NeuroPore™ is a non-proteolytic permeabilization and blocking reagent developed for use with the NeuroTACS™ In Situ Apoptosis Detection Kit (Cat# 4820-30-K). Many tissues of the central nervous system are fragile and do not tolerate harsh proteolytic treatment. Neuro- Pore™ provides a gentle method for the permeabilization of tissues prior to in situ labeling. In addition, NeuroPore™ is an ideal antibody diluent for use in double labeling experiments: antigenic determinants are retained during permeabilization and can be detected using standard immunohistochemical techniques in conjunction with in situ detection of apoptosis.

Cytonin™

Cytonin™ offers an alternative to Proteinase K for permeabilizing cells and tissues prior to labeling using any of the TACS® In Situ Apoptosis Detection kits. Cytonin™ is a protease free, saponin-based buffer designed specifically for in situ detection of apoptosis. Cytonin™ should be used when protease treatment must be avoided. For example, it may be used for tissues with low cellularity or little connective tissue that do not withstand protease treatment. Cytonin IHC™ (see below) is recommended for double labeling experiments for the detection of DNA fragmentation in conjunction with immunohistochemistry on antigens that are sensitive to proteolysis. 157 Cytonin-IHC™

Cytonin-IHC™ is a non-proteolytic, saponin-based permeabilization and blocking reagent designed specifically for double labeling experiments using TACS® In Situ Apoptosis Detection kits, and a primary antibody of your choice. Cytonin IHC™ is used as a convenient time- saving antibody diluent for immunohistochemistry, allowing you to simultaneously perform your primary antibody incubation and permeabilization of your sample for in situ detection of apoptosis. In fact, use Cytonin IHC™ as a diluent for your secondary antibody too. Slides and Coverslips Treated Glass Microscope Slides

ORDERING INFORMATION These slides are specially treated to promote electrostatic adhesion of your tissue or cell samples. The slides are frosted on one end allowing you to label the sample with a pencil or 4800-30-01 Proteinase K, 30 µl histology pen for clear identification. The slides are precleaned and ready for use. 4820-60-01 Treated Glass Microscope Slides with 3 Ring Hydrophobic Barrier NeuroPore™, 2 x 5 ml 4876-05-02 These slides have the same coating as our treated glass microscope slides (Cat# 4861-100) Cytonin™, 2 x 5 ml with an added 3 ring hydrophobic barrier allowing you to process 3 different samples easily 4878-05-01 on one slide. The barrier promotes better sample coverage and conserves reagents. Also, Cytonin-IHC™, 5 ml the barrier makes the task of drying suspension cells onto slides as easy as pipetting. 4878-05-02 Cytonin-IHC™, 2 x 5 ml 4861-100 Treated Glass Microscope Slides, 100 each 4864-100 Treated Glass Microscope Slides with 3 Ring Hydrophobic Barrier, 100 each 4876-05-01 Cytonin™, 5 ml

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APOPTOSIS DETECTION

Apoptosis Research Accessories

Slides and Coverslips Hydrophobic Coverslips

These coverslips are made of a specially treated, rigid plastic that produces a hydrophobic surface, assuring rapid and even distribution of reagents over the entire sample being labeled. The coverslips should be used to conserve reagents when larger sample areas are being labeled. Unlike glass coverslips that may not wet evenly and may be too heavy to allow sufficient reagent to contact the sample, these coverslips float easily on small volumes of labeling reagents. Unlike wax film or other commercially available coverslips, these coverslips are rigid, preventing uneven distribution of labeling solutions. The coverslips are packaged in rows of 5 with a protective film coating.

Glass Coverslips

These coverslips are optically clear glass ideal for mounting onto your specimens prior to viewing. The coverslips are clean and ready for use. The 22 x 60 mm size assures complete coverage when using our 3 ring barrier slides (Cat# 4864-100) or processing large samples. These coverslips may be used with Trevigen Mounting Medium (Cat# 4865-25) or Trevigen Fluorescence Mounting Medium (Cat# 4866-20). Streptavidin Conjugate

APOPTOSIS RESEARCH ACCESSORIES Trevigen offers a number of streptavidin conjugates to allow flexibility in both in situ labeling, and our flow cytometric-based assays (e.g. FlowTACS™). Optimal concentrations range from 1:50 158 dilutions to 1:1000 dilutions, depending upon application. The table below summarizes the characteristics of the fluorescent conjugate.

Abs. Max Em. Max Streptavidin-AMCA 345 nm 445 nm "Blue"

Streptavidin-FITC 492 nm 520 nm "Green"

ORDERING INFORMATION

4800-30-06 Streptavidin-HRP, 30 µl 4800-30-14 Streptavidin-FITC, 30 µl 4867-100 Hydrophopic Coverslips, 100 each 4862-10 Glass Coverslips (22 X 60 mm), 10 X 1 oz. 4887-100-03 Streptavidin-AMCA, 100 µl

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APOPTOSIS DETECTION

Product Use Hints, Tips, and Protocols

Annexin V Conjugates The use of annexin V conjugates to detect apoptotic cells is one of the most simple and rapid biochemical detection methods for apoptosis. When combined with the power of flow cytometry, quantitative data on a large cell population can be generated rapidly (within minutes) and accurately.

ANNEXIN V-FITC The Annexin V-FITC conjugate is most appropriate for analysis of apoptosis in cells in suspension when long term preservation of the sample is not required. Annexin V-FITC is added to suspension cells, in the presence of calcium, which can be assayed directly by flow cytometry. Extensive washes or incubations are not required. Annexin V positive cells are typically one to two logs higher in relative APOPTOSIS PRODUCT USE fluorescence intensity than unlabeled cells (i.e. healthy cells). The optimal time post induction of apoptosis for Annexin V labeling can vary by cell type, so some optimization may be required for optimal results. The addition of propidium iodide to annexin V-labeled samples allows identification of late apoptotic, and dead or necrotic cells with compromised plasma membranes. To aid in setting up the flow cytometer, a positive sample can be generated by fixing the cell sample in 3% formaldehyde for 20 minutes prior to performing the assay. Fixed cells take up annexin V and propidium iodide intracellularly, however, the profile of fixed and then labeled cells is slightly different to those of unfixed cells and often have a lower fluorescent signal. Since propidium iodide is taken up by all fixed cells, it can no longer be used to distinguish between early apoptoic and necrotic cells in this application. For optimal results, the use of unfixed cells is recommended when evaluating experimental samples. To evaluate the specificity of Annexin-V labeling (i.e. background), the assay can be performed in the presence of a chelating agent. Specific Annexin-V binding is calcium dependent, any binding that occurs in the absence of calcium is non-specific.

ANNEXIN V-BIOTIN 159 The Annexin V-Biotin conjugate provides a variety of options to the user. In most cases, FITC is quenched when treated with fixatives used to preserve cell samples. The need to fix cell samples may be important in extended time course experiments, coordinating the experiment with equipment availability, and the biohazardous state of the sample. Annexin V-Biotin has the advantage of stability following fixation. For detection, any appropriate streptavidin conjugate may be used, thus providing options in selection of the fluorophore. The option to use alternative fluorophores is important when other markers, e.g. cell surface antigens, are being detected. For detection of apoptosis using Annexin V-biotin, cells are first incubated in the presence of calcium with Annexin V-Biotin, washed, and then the cells may be fixed if desired. A streptavidin-conjugate, e.g. streptavidin-PE, is then added and the sample is analyzed by flow cytometry. A single wash after incubation with the streptavidin conjugate is usually all that is required to reduce any non-specific binding. Since Annexin V-Biotin is bound to the cell surface, there is no limitation to the size of the streptavidin conjugate, or access of the streptavidin conjugate to the biotin, unlike the detection of intracellular markers when large fluorophores such as PE have to be avoided. Additionally, all fixed cells will take up propidium iodide, rendering this reagent inappropriate for discriminating between fixed necrotic and apoptotic cells. To determine the level of background labeling by Annexin V-biotin, labeling can be performed in the presence of a chelating agent. Since Annexin-V binding is calcium dependent, any binding that occurs in the absence of calcium is non-specific.

Bcl-2 Family of Antibodies The Bcl-2 family of monoclonal antibodies was developed using synthetic peptides corresponding to functional domains within the Bcl-2, Bax and Bcl-XL proteins. The immunogens were designed either for species-specific recognition or to have reactivity among multiple species. These antibodies are purified from mouse ascites and provided in phosphate buffered saline with a preservative. These purified antibodies have proven extremely useful in delineating important interactions between the family members, including dimer formation.

WESTERN BLOT Trevigen’s antibodies have been qualified for use in Western blotting. Expression levels of the Bcl-2 family of proteins may be low, and are not necessarily upregulated during apoptosis. If expression levels are expected to be low, partial purification, e.g. by immunoprecipitation, may be needed to generate a strong signal in a Western blot. A simple procedure for the preparation of cell lysates for SDS-PAGE and Western blotting for using any of Trevigen’s antibodies is provided. 1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:23 PM Page 164

APOPTOSIS DETECTION

Product Use Hints, Tips, and Protocols

Trevigen recommends using PVDF membrane instead of nitrocellulose for Western blotting since it consistently produced a higher signal-to-noise ratio. Titration of the antibody is very important, and both the primary and secondary antibody should be tested at several dilutions to obtain optimal visualization.

Preparing Cell Lysates 1.1 For adherent cells or tissues, generate a single cell suspension using standard methods. 1.2 Centrifuge cells at 200 x g for 3 min, aspirate supernatant, resuspend cell pellet in PBS and count cells using a hemocytometer. 1.3 Centrifuge the cell suspension at 200 x g for 3 min. 1.4 Aspirate PBS and resuspend the cell pellet in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP40, 0.5% Sodium Deoxycholate, 1 mM PMSF, 1X protease inhibitor cocktail) at a final concentration of 2 x 106 - 2 x 107 cells per ml. 1.5 Incubate on ice for 10 min. 1.6 Pipette solution up and down a few times and transfer it into microtube. 1.7 Centrifuge for 10 min at 14,000 x g at 4oC. 1.8 Recover the supernatant and add an equal volume of 2X Tris-Glycine SDS gel loading buffer.

SDS-PAGE and Western blot 1. Load samples onto a 4-20% Tris-Glycine SDS-PAGE gel and perform electrophoresis for APOPTOSIS PRODUCT USE the recommended time as instructed by the manufacturer. After electrophoresis is complete, transfer proteins onto a PVDF membrane according to manufacturer’s instructions for the 160 transfer apparatus. 2. Rinse the PVDF membrane with dH2O and verify transfer by staining with Ponceau Red Solution. 3. Rinse the membrane with TBS-T (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1%Tween-20) and incubate for 1 hour at room temperature in Blocking Solution (5% (w/v) nonfat dry milk in TBS-T). 4. Dilute the primary antibody in Blocking Solution and incubate membrane for 1 hour at room temperature or overnight at 4˚C. 5. Rinse the membrane 3 times for 5 min each, with TBS-T. 6. Incubate the membrane for 1 hour at room temperature with the HRP-conjugated secondary antibody, diluted in Blocking Solution. 7. Rinse the membrane 4 times for 10 min with TBS-T. 8. Prepare the chemiluminescent solution, immediately pour it onto the membrane and incubate for 1 min. 9. Decant the excess amount of chemiluminescent solution, but do not let the membrane dry, wrap the membrane in plastic wrap. 10. In a dark room overlay the membrane onto film in a film cassette, and proceed with developing.

Immunohistochemistry Trevigen has not qualified the Bcl-2 family of antibodies for immunohistochemistry. However, there are published reports of successful immunolabeling with anti-Bax antibodies YTH-2D2 and YTH-6A7 (e.g. Neuchushtan, A., C.L. Smith, Y.-T. Hsu, and R.J. Youle. 1999. Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO. J. 18:2330-2341; Utsunomiya K et al. 2004 Crossreaction with an Anti-Bax Antibody Reveals Novel Multi-endocrin Cellular Antigen. J Histochemistry and Cytology 52:805-812) and with the anti-Bcl-XL antibody YTH-2H12 (e.g. Chu, W.-S., N.S.I. Aguilera, M.Q. Wei, and S.L. Abbondanzo. 1999. Antiapoptotic marker, Bcl-XL, expression on Reed-Sternberg cells of Hodgkin’s disease using a novel monoclonal marker, YTH-2H12. Hum. Pathol. 30:1065-1070; Inomata A et al. 2002 5-Fluorouracil-induced intestinal toxicity: what determines the severity of damage to murine intestinal crypt epithelia? Toxicology Letters 133:231-240).

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APOPTOSIS DETECTION

Product Use Hints, Tips, and Protocols

Protocols for In Situ Apoptosis Detection: Decalcification of Bone and Joint Tissues For in situ labeling of apoptotic cells in bone and cartilage, a gentle method for the decalcification is required. Treatment of tissue will vary with size, porosity, and type of tissue, therefore the following is provided as a guideline only:

Clean muscle away from bone carefully. Do not scrape the outside edge of the bone. For optimal labeling, paraformaldehyde is frequently used. Tissue samples should be fixed in 4% paraformaldehyde in PBS buffer (10% neutral buffered formalin or 2-4% formaldehyde prepared APOPTOSIS PRODUCT USE in PBS buffer may also be used) for 8 to 12 hours with one or two changes of paraformaldehyde solution. It may be necessary to mechanically (GENTLY!) separate the joint with dissection tools while in fixative to facilitate fixation throughout the entire tissue.

Once the tissue is fixed, it should be placed into 10 volumes EDTA decalcification solution for up to 3 weeks at 4°C, in order to process bone and cartilage samples for standard histological techniques. It will be necessary to change the EDTA solution several times during this period. Decalcification should be allowed to continue until the tissue is pliable, like soft rubber, and times for this will need to be determined. The tissue should be sectionable at this point. If further decalcification is required, it is possible to further decalcify sectioned tissues on slides.

The tissue should be placed into an increasing series of ethanol (70%, 95%,100%) and then xylene prior to embedding in paraffin. If necessary the tissue can be held in 70% ETOH for several days before completing the dehydration procedure. Sections should be cut at 5-10 µm, and floated onto 161 glass slides treated for electrostatic adherence. Sections should be dried at 45˚C for up to one hour or at room temperature overnight. Do not bake the slides. Slides are ready for labeling at this point.

Decalcification Solution: 14% EDTA in 1X PBS

Note: Paraformaldehyde fixation, prior to embedding, is reversible when samples are stored in liquids for long periods. Thus, include 2.5% Paraformaldehyde in the decalcification solution to maintain fixation.

Reduction of Cross-Linking in Over-Fixed Tissues Samples that have been over-fixed with paraformaldehyde or formaldehyde (formalin) can often be used for in situ apoptosis detection following reduction of cross-linking. This method is commonly performed for immunohistochemistry, however, the citrate buffer-microwaving technique is not fully compatible with the DNA within tissues. The elevated temperatures used can cause a significant pH drop in the citrate buffer resulting in DNA degradation. The method described below helps to minimize the DNA damage although DNA damage can still induced.

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APOPTOSIS DETECTION

Product Use Hints, Tips, and Protocols

Prepare the following:

Stock solutions: A. Solution of citric acid 0.1 M 21.01 g Citric acid, monohydrate (C6H8O7H2O) 1000.0 ml Distilled H2O

B. Solution of sodium citrate 0.1 M 29.41 g Trisodium citrate dihydrate (C6H5Na3O72H2O) 1000.0 ml Distilled H2O

Working solution (0.01M Citrate Buffer, pH 6.0): 9.0 ml Solution A 41.0 ml Solution B 450.0 ml Distilled H2O

1. Place solution in a Coplin jar and heat up to 80˚C. (Microwaving of the slides is not recommended). 2. Immerse slides in solution and cool slowly to room temperature. APOPTOSIS PRODUCT USE

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DNA Damage and Genomic Instability STEM CELL PRODUCTS

Cancer Cell Behavior

Apoptosis Detection

STEM CELL PRODUCTS

Oxidative Stress

Molecular Biology Reagents STEM CELL PRODUCTS STEM INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:23 PM Page 168

STEM CELL PRODUCTS

Overview

At Trevigen we are proud of our reputation for supplying the highest quality reagents and kits to the research community. As part of our ongoing effort to offer new products and find new applications for our existing reagents we now offer tools and information to help facilitate stem cell research. Trevigen now provides rat mesenchymal stem cells and kits for their differentiation into Adipocytes and Osteoblasts. For culturing and examining the properties of human embryonic stem cells as well as cancer stem cells, Trevigen’s murine derived and human derived BME is currently being used by scientists internationally. Please do not hesitate to call us for more information with regard to the use of BME for stem cell research.

Please check our website for the latest product additions and product information. STEM CELL PRODUCTS OVERVIEW

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STEM CELL PRODUCTS

Basement Membrane Extract (BME) Cultrex® Stem Cell Qualified Basement Membrane Extract (BME), PathClear®

Cultrex® Stem Cell Qualified Basement Membrane Extract (BME), PathClear® is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor. The extract gels at 37 °C to form a reconstituted basement membrane. Mainly comprised of laminin, collagen

IV, entactin, and heparin sulfate proteoglycan Cultrex® Stem Cell Qualified BME has been shown STEM CELL QUALIFIED BME PathClear to provide an effective feeder-free surface for the attachment of and maintenance of human embryonic stem cells in a pluripotent state, thereby enabling its use for growth promotion or for study of stem cell differentiation.

Specifications:

CONCENTRATION:

12–18 mg/ml

SOURCE:

Murine Engelbreth-Holm-Swarm (EHS) tumor. ®

STORAGE BUFFER: 165 Dulbecco’s Modified Eagle’s medium containing 10 µg/ml gentamycin sulfate, without phenol red.

STORAGE/STABILITY:

Material Qualification: H9 human embryonic stem cells after four passages on Cultrex Stem Cell Qualified BME bind DAPI (A) and maintain FUNCTIONAL ASSAY: expression of the non-differentiated stem cell markers Oct-4 (B) and Nanog (C). • Promotes the attachment of H9 human embryonic stem cells. Images courtesy of the Yanik lab, MIT (www.rle.mit.edu/bbng) • Effectively maintains human embryonic stem cells in a pluripotent state as evidenced by intracellular stains for the stem cell markers Oct-4 and Nanog.

STERILITY TESTING:

• No bacterial or fungal growth detected after incubation at 37 °C for 14 days following USP XXIV Chapter 71 sterility test. ORDERING INFORMATION • Negative by PCR test for: mycoplasma, 17 bacterial and virus strains typically included in 3434-001-02 mouse antibody production (MAP) testing, plus 13 additional murine infectious agents Cultrex® Stem Cell Qualified BME, including LDEV, for a total of 31 organisms and viruses. Phenol Red Free, Reduced Growth ® Factor, PathClear , 1 ml • Endotoxin concentrations ≤ 8 EU/ml by LAL assay 3434-005-02 Cultrex® Stem Cell Qualified BME, REFERENCES: Phenol Red Free, Reduced Growth ® Factor, PathClear , 5 ml 1. Angel, M. and M. F. Yanik. 2010. Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins. PLoS ONE. 5(7):e11756. 1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:23 PM Page 170

STEM CELL PRODUCTS

Rat Mesenchymal Stem Cells

Mesenchymal Stem Cells (MSC), also known as marrow stromal cells, are a self-renewing population of multipotent cells present in bone marrow and many other adult tissues. MSC can be isolated from bone marrow by adherence to plastic, and can differentiate into multiple lineage-specific cells that form bone, fat, cartilage, muscle, neuronal cells and tendon. (Figure 1) Bone Due to their multilineage potential, they can be useful tools for a wide range of therapeutic and basic research, including transplantation studies and studies examining the repair of cardiac

Adipose Tendon tissue, bone, cartilage, and tendons often using 3-D matrices. The generation of dependable information that can be shared between laboratories requires a consistent, highly purified

Mesenchymal source of MSC that have maintained their multipotency, as demonstrated by their ability to Stem Cells differentiate to different tissue types. Isolation of MSC is both a time and resource consuming process which can now be eliminated. A readily available and qualified source of cells will Muscle Marrow Stroma substantially decrease MSC preparation time required prior to experimentation.

6 Cartilage Trevigen's RMSC are provided as a frozen ampoule containing 1 x 10 passage 3 cells that are greater than 99% pure. These cells can be maintained in an undifferentiated state when grown in Trevigen's qualified RMSC Medium supplemented with RMSC qualified FBS or induced to differentiate into adipogenic or osteogenic phenotype when growth media are Figure 1: Multilineage potential of Adult Rat Mesenchymal supplemented with reagents contained in Trevigen's differentiation kits (see page 167,168). Stem Cells Trevigen's RMSC can undergo 10 doublings without alteration in cell morphology or differentiation potential.

FEATURES:

RAT MESENCHYMAL STEM CELLS RAT • Primary Mesenchymal Stem Cells isolated and purified from Rat Bone Marrow. • Qualified medium and fetal bovine serum to support undifferentiated growth 166 of rat mesenchymal stem cells. APPLICATIONS: Therapeutic and basic research, including transplantation studies and studies examining the repair of cardiac tissue, bone, cartilage, and tendons often using 3-D matrices.

STORAGE: See Below

Catalog # Component Storage 5000-001-01 Cultrex® Rat Mesenchymal Stem Cell Liquid Nitrogen 5000-001-K Cultrex® Rat Mesenchymal Stem Cell Starter Kit Cultrex® Rat Mesenchymal Stem Cell Liquid Nitrogen Cultrex® Qualified RMSC Medium 4°C Cultrex® Qualified RMSC FBS -20°C 5000-001-R Cultrex® Rat Mesenchymal Stem Cell Replenisher Kit Cultrex® Qualified RMSC Medium 4°C Cultrex® Qualified RMSC FBS -20°C

ORDERING INFORMATION RELATED PRODUCTS: 5000-001-01 • Cultrex® Adipogenic Differentiation Kit. ® Cultrex Rat Mesenchymal Stem Cell, • Cultrex® Osteogenic Differentiation Kit. 1 vial (1x 106) 5000-001-K REFERENCES: Cultrex® Rat Mesenchymal Stem Cell 1. Phinney DG, Prockop DJ. 2007. Concise Review: Mesenchymal Stem/Multipotent Stromal Starter Kit; Cells: The State of Transdifferentiation and Models of Tissue Repair-Current Views. Stem Cells ® Cultrex Rat Mesenchymal Stem Cell; 25:2896-2902. ® Cultrex Qualified RMSC Medium; 2. Kolf CM, Cho E, Tuan RS. 2007. Biology of Adult Mesenchymal Stem Cells: Regulation of Cultrex® Qualified RMSC FBS, 24 Wells Niche, Self-Renewal and Differentiation. Arthritis Research and Therapy 9:204. 5000-001-R 3. Javazon EH, Colter DC, Schwarz EJ, Prockop DJ. 2001. Rat Marrow Stromal Cells are More Cultrex® Rat Mesenchymal Stem Cell Sensative to Plating Density and Expand More Rapidly from Single-Cell-Derived Colonies than Replenisher Kit; Human Marrow Stromal Cells. Stem Cells 19:219-225. ® Cultrex Qualified RMSC Medium; 4. Li Yi, McIntosh K, Chen J, Zhang C, Gao, Q, Borneman J, Ragniski K, Mitchell J, Shen L, Zhang Cultrex® Qualified RMSC FBS, 500/50 ml J, Lu D, Chopp M. 2006. Allogeneic bone marrow stromal cells promote glial-axonal remodeling without immunologic sensitization after stroke in rats. Experimental Neurology 198:313-25. 1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:23 PM Page 171

STEM CELL PRODUCTS

Cultrex® Mesenchymal Stem Cell Adipogenic Differentiation Kit

Adipogenic Differentiation Adipogenesis is the process by which fat is formed.(1) Fat formation plays a role in obesity, cardiovascular disease, and metabolic disorders.(1) It also has a role in the maintenance of the bone marrow cavity. Adipocytes are derived from mesenchymal stem cells (MSC), which are a self-renewing population of multipotent cells present in bone marrow and many other adult tissues that can differentiate into multiple lineage-specific cells that form bone, fat, cartilage, muscle and tendon.(2-5) Differentiation of primary cells, specifically MSC, into adipocytes can be a useful tool for understanding the mechanisms involved in adipogenesis

leading eventually to interventions designed to prevent obesity. ADIPOGENIC DIFFERENTIATION

Trevigen’s adipogenic differentiation kit follows traditional methods of adipogenic differentiation,(6) by growing MSC in medium supplemented with insulin, isobutyl methyl xanthine (IBMX), indomethacin and dexamethasone. The kit will induce adipogenic differentiation of MSC within Trevigen cultured RMSC induced into adipogenic differentiation using Trevigen Adipogenic Differentiation 14-17 days. Adipogenic differentiation is detected by staining with Oil Red O. Oil Red O will Kit. 14 day cultures were stained with Oil Red O to stain lipid containing vacuoles in mesenchymal stem cells that have undergone visualize lipid droplets, a marker of adipogenic differentiation. adipogenic differentiation.

FEATURES: Optimized reagents for the differentiation of Mesenchymal Stem Cells into Adipocytes. Sufficient reagents to differentiate one 24 well plate and stain two 24 well plates. The amount of differentiation can be quantified.

APPLICATIONS: Tool for understanding mechanisms involved in adipogenesis eventually leading to interventions designed to prevent obesity.

STORAGE: See Below. 167

Catalog # Component Size Storage 5010-024-01 Dexamethasone 12 µl -20°C 5010-024-02 IBMX 65 µl -20°C 5010-024-03 Insulin 110 µl 4°C 5010-024-04 Indomethasin 330 µl -20°C 5010-024-05 Oil Red O 15 ml RT

RELATED PRODUCTS: Cultrex® Rat Mesenchymal Stem Cells, Cultrex® Rat Mesenchymal Stem Cell Starter Kit, Cultrex® Rat Mesenchymal Stem Cell Replenisher, Cultrex® Osteogenic Differentiation Kit.

REFERENCES: 1. Kiess W, Petzold S, Topfer M, Garten A, Bluher, Kapellen T, Korner A, Kratzsch J 2008 Adipocytes and Adipose Tissue. Best Practice and Research Clinical Endocrinology and Metabolism 22(1):135-153. 2. Phinney DG AND Prockop DJ 2007. Concise Review: Mesenchymal Stem/Multipotent Stromal Cells: The State of Transdifferentiation and Models of Tissue Repair-Current Views. Stem Cells 25:2896-2902 3. Kolf CM, Cho E, Tuan RS. 2007. Biology of Adult Mesenchymal Stem Cells: Regulation of Niche, Self-Renewal and Differentiation. Arthritis Research and Therapy 9(1): 204-214 4. Javazon EH, Colter DC, Schwarz EJ, Prockop DJ. 2001. Rat Marrow Stromal Cells are More Sensative to Plating Density and Expand More Rapidly from Single-Cell-Derived Colonies than Human Marrow Stromal Cells. Stem Cells 19:219-225 5. Li Yi, McIntosh K, Chen J, Zhang C, Gao, Q, Borneman J, Ragniski K, Mitchell J, Shen L, Zhang J, Lu D, Chopp M. Allogeneic bone marrow stromal cells promote glial-axonal remodeling without immunologic sensitization after stroke in rats. Experimental Neurology ORDERING INFORMATION 198(2):313-25. 6. Reger RL, Tucker AH, Wolfe MR. 2008. Differentiation and Characterization of Human 5010-024-K MSCs. In Prockop DJ, Phinney DG, Bunnell BA (eds) Methods in Molecular Biology Mesenchymal Stem Cell Adipogenic Mesenchymal Stem Cells: Methods and Protocols vol 449. Humana Press, Totawa, NJ, Differentiation Kit, 24 Wells USA pp 93-107.

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STEM CELL PRODUCTS

Cultrex® Mesenchymal Stem Cell Osteogenic Differentiation Kit

Control Mesenchymal Stem Cells, a self-renewing population of multipotent cells, can differentiate into osteogenic cells 1,2 that will deposit and mineralize extracellular matrix proteins, 2,3 which is required for the formation of new bone. Measuring the amount of mineralized matrix is an established method for evaluating osteogenic differentiation.2

Trevigen’s Mesenchymal Stem Cell Osteogenic Differentiation Kit contains reagents optimized to direct mesenchymal stem cells grown on Cultrex® Rat Collagen I4 to undergo osteogenic differentiation in a defined growth medium supplemented with ascorbic acid, β-glycerol phosphate, dexamethasone within 14 days. Osteogenic differentiation is detected by staining with Alizarin Red S5. Alizarin red will stain the calcium deposits that occur when differentiated mesenchymal stem cells mineralize the extracellular matrix.5

Trevigen RMSC cultured in Trevigen’s qualified medium and serum for 14 days. Stained with Alizarin Red S to visualize The differentiation of mesenchymal stem cells into osteogenic cells can be a useful tool for calcium deposits, a marker of osteogenic differentiation. understanding mechanisms involved in bone formation. It is also used for identifying potential treatments for osteoporosis, bone repair and genetic disorders such as osteogenesis imperfecta. 3

FEATURES: Optimized reagents for the differentiation of Mesenchymal Stem Cells into osteoblasts. Quantifiable, reagents to quantify amount of Alizarin Red Staining included. Sufficient reagents to differentiate one 24 well plate.

OSTEOGENIC DIFFERENTIATION Sufficient reagents are provided to coat, stain and quantify two 24 well plates. Osteogenic Differentiation 168 APPLICATIONS: Tool for understanding mechanisms involved in bone formation and for identifying potential treatments for osteoporosis, bone repair and genetic disorders such as osteogenesis imperfecta.

STORAGE: See Below.

Catalog # Component Size Storage 5010-024-01 Dexamethasone 12 µl -20°C 5011-024-01 Ascorbic Acid 1.2 ml 4°C 5011-024-02 β-Glycerol Phosphate 1.2 ml 4°C 3440-001-01 Cultrex® Rat Collagen I 500 µl 4°C Trevigen cultured RMSC induced into osteogenic differen- 5011-024-03 Alizarin Red S 25 ml RT tiation using Trevigen Osteogenic Differentiation Kit. 14 day 5011-024-04 Stain Solubilization Solution 35 ml RT cultures were stained with Alizarin Red S to visualize calcium deposits, a marker of osteogenic differentiation. RELATED PRODUCTS: Cultrex® Rat Mesenchymal Stem Cells, Cultrex® Rat Mesenchymal Stem Cell Starter Kit, Cultrex® Rat Mesenchymal Stem Cell Replenisher, Cultrex® Adipogenic Differentiation Kit.

REFERENCES: 1. Satija NK, Gurudutta GU, Sharma S, Afrin F, Gupta P, Verma K, Singh VK, Tripathi RP. 2007 Mesenchymal Stem Cells: Molecular Targets for Tissue Engineering. Stem Cells and Development 16:7-23. 2. Jones JL, Scotting P, Sottile V. 2007. Adult Mesenchymal stem cell: Differentiation potential and therapeutic applications. J Postgrad Med 53(2): 121-127. 3. Li F, Wang, X, Niyibizi C. 2007. Distribution of Single-Cell Expanded Marrow Derived Progenitors in a Developing Mouse Model of Osteogensis Imperfecta Following Systemic Treatment. Stem Cells. 25:3183-3193. 4. Salasznyk RM, Williams WA, Boskey A, Batorsky A, Plopper GE. 2004. Adhesion to Vitronectin and Collagen I Promotes Osteogenic Differentiation of Human Mesenchymal ORDERING INFORMATION Stem Cells. J. Biomedicine and Biotechnology 2004:24-34. 5. Reger RL, Tucker AH, Wolfe MR. 2008. Differentiation and Characterization of Human 5011-024-K MSCs. In Prockop DJ, Phinney DG, Bunnell BA (eds) Methods in Molecular Biology Mesenchymal Stem Cell Osteogenic Mesenchymal Stem Cells: Methods and Protocols vol 449. Humana Press, Totawa, NJ, USA Differentiation Kit, 24 Wells pp 93-107.

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DNA Damage and Genomic Instability OXIDATIVE STRESS

Cancer Cell Behavior

Apoptosis Detection

Stem Cell Products

OXIDATIVE STRESS

Molecular Biology Reagents OXIDATIVE STRESS OXIDATIVE INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:24 PM Page 174

OXIDATIVE STRESS

Overview

Oxidative stress, which leads to DNA damage, is a primary driver leading to the accumulation of mutations which occurs in living organisms. The estimated frequency of oxidatively-induced lesions is approximately 104/cell/day. The most extensively investigated, and most abundant DNA lesion generated following exposure to hydroxyl radicals,is 8-hydroxy-2’-deoxy-guanosine (8-oxo-dG). Trevigen now offers two products to enable investigators to monitor changes in 8-oxo-dG levels: a high throughput (HT) 8-oxo-dG ELISA Kit, and 8-oxo-dG monoclonal antibody for immunohistochemical, and immunocytochemical detection.

Trevigen also offers research kits targeting biomarkers of oxidative stress. These kits employ well-established methods to assay for superoxide dismutase (SOD), glutathione reductase, glutathione peroxidase, or glutathione, which directly and indirectly measure cellular defensive responses against oxidative stress. Cellular responses to oxidative stress are of interest to investigators of a wide variety of human diseases such as: cancer, diabetes, atherosclerosis, stroke, Alzheimer’s, many auto-immune diseases, and aging.

Please check our website for the latest product additions and product information.

Oxidative Stress Assay Kits OXIDATIVE STRESS OVERVIEW OVERVIEW OXIDATIVE

Kit Catalog # Application(s) Detection Method Format Sensitivity Time* Page 1700 0 Super Oxide Dismutase Detection of SOD1 7501-500-K [(Cu/Zn)-SOD], SOD2 WST-1 96-well Plate 0.1-10 unit of SOD 10 minutes HT Super Oxide 2+ 173 Dismutase Kit [(Mn )-SOD], and/or (A450) 0.5 to 50 µg of Iysate (per plate) SOD3 [Fe2+)-SOD] Detection of SOD1 Super Oxide 7500-100-K [(Cu/Zn)-SOD], SOD2 NBT-diformazan Cuvette 0.1-10 unit of SOD 5 minutes Dismutase Kit [(Mn2+)-SOD], and/or (A550) (per sample) 174 SOD3 [Fe2+)-SOD] Glutathione Related Detection of Total HT Total Glutathione 7511-100-K Reduce, and Oxidize DTNB 96-well Plate 12.5-100 pmoles of 10 minutes 175 Assay Kit Glutathione (A405) GSSG (per plate) Detection of 10 minutes 7512-100-K Glutathione 96-well Plate (per plate) HT Glutathione NADPH N/A, Cycling 176 Peroxidase Assay Kit Peroxidase (A340) reaction. Cuvette 10 minutes (per sample) Detection of Glutathione Reductase 7510-100-K NADPH Cuvette 1-10 mUnits/ml 6 minutes 178 Glutathione (A340) (per sample) Assay Kit Reductase Detection of HT Glutathione 7513-500-K NADPH 96-well Plate 1-10 mUnits/ml 10 minutes Glutathione 0.5 to 50 µg of Iysate (per plate) 177 Reductase Assay Kit Reductase (A340) 8-oxo-dG Detection of 8-oxo-dG in urine, serum and TMB 2.5 hours 171 8-oxo-dG Elisa kit 4370-096-K saliva samples (A450) 96-well Plate 0.59 ng/ml (per plate)

*Actual times may vary depending upon the number of samples being prepared and assayed.

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OXIDATIVE STRESS

HT 8-oxo-dG ELISA Kit

Typical 8-oxo-dG Standard Curve 8-hydroxy-2’-deoxyguanosine (8-oxo-dG) is a modified nucleoside, which is the most commonly studied and detected by-product of DNA damage that is excreted following DNA repair. The presence of 8-oxo-dG and its analogs, 8-hydroxyguanosine and 8-hydroxyguanine in the urine, is associated

2.5 with many degenerative disease states in which reactive oxygen species (ROS) contribute to pathogenesis. 8-oxo-dG, a biomarker of oxidative stress, has been employed as an ROS indicator 2 in many studies on a wide variety of diseases.

1.5 Trevigen’s 8-oxo-dG ELISA (enzyme-linked immunosorbent assay) is a fast and sensitive, competitive 1 immunoassay for the detection and quantitation of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) in Absorbance (450nm) 0.5 saliva, urine and serum samples. This kit features a 96 strip well plate pre-bound with 8-oxo-dG, an 8-oxo-dG monoclonal antibody, an enzyme-labeled secondary antibody, detection substrates and 0

0 0.5 1 1.5 2 buffer to provide a complete, robust assay flexible for your experimental design. HT 8-oxo-dG ELISA KIT Log 8-oxo-dG Concentration (ng/mL) FEATURES: • Colorimetric format • High throughput 96 test size • Standard curve has a range of 0.94 - 60 ng/ml • Sensitive to 0.59 ng/ml

APPLICATIONS: For the detection and quantitation of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) in urine, serum and saliva samples.

COMPONENTS: Catalog # Component Size 4370-096-01 8-oxo-dG Immunoassay Plate 96 well plate 171 4370-096-02 8-oxo-dG Standard 25 µl 4370-096-03 Sample Diluent 50 ml 4370-096-04 20x Wash Buffer 100 ml 4370-096-05 Anti-8-oxo-dG 25 µl 4370-096-06 Antibody Diluent 6 ml 4370-096-07 Anti-Mouse IgG HRP Conjugate 25 µl 4370-096-08 HRP Conjugate Diluent 12 ml 4370-096-09 TMB Substrate 12 ml 4370-096-10 Stop Solution 2 11 ml

STORAGE: All reagents are stable as supplied at 4°C, except the 8-oxo-dG standard, which should be stored at -20°C. For optimum storage, the 8-oxo-dG standard should be aliquoted into smaller portions and stored at -20°C to avoid repeated freeze/ thaw cycles.

Unused wells of the 8-oxo-dG Immunoassay Plate should be resealed with desiccant in the foil pouch provided and stored at 4°C until the kit expiration date.

REFERENCES: 1. Evans, M. D., Dizdaroglu, M., and Cooke, M. S. (2004) Mutat. Res. 567: 1-61. 2. Sancar, A., Lindsey-Boltz, L. A., Unsal-Kacmaz, K., and Linn, S. (2004) Annu. Rev. Biochem. 73: 39-85. 3. Chiou, C. C., Chang, P. Y., Chan, E. C., Wu, T. L., Tsao, K. C., and Wu, J. T. (2003) Clin. Chim. Acta. 334: 87-94. 4. Trzeciak, A. R., Nyaga, S. G., Jaruga, P., Lohani, A., Dizdaroglu, M., and Evans, M. K. (2004) Carcinogenesis 25: 1359-1370. 5. Brown, R. K., McBurney, A., Lunec, J., and Kelly, F. J. (1995) Free Radic. Biol. Med. 18: 801-806. 6. Tsuboi, H., Kouda, K., Takeuchi, H., Takigawa, M., Masamoto, Y., Takeuchi, M., and Ochi, H. (1998) Br. J. Dermatol. 138: 1033-1035. ORDERING INFORMATION 7. Rall, L. C., Roubenoff, R., Meydani, S. N., Han, S. N., and Meydani, M. (2000) J. Nutr. Biochem. 11: 581-584. 4370-096-K 8. Lezza, A. M., Mecocci, P., Cormio, A., Beal, M. F., Cherubini, A., Cantatore, P., Senin, U., and HT 8-oxo-dG ELISA Kit, 96 Wells Gadaleta, M. N. (1999) FASEB J. 13: 1083-1088. 9. Alam, Z. I., Jenner, A., Daniel, S. E., Lees, A. J., Cairns, N., Marsden, C. D., Jenner, P., and Halliwell, B. (1997) J. Neurochem. 69: 1196-1203. 1-800-873-8443 • www.trevigen.com INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:24 PM Page 176

OXIDATIVE STRESS

Anti-8-oxo-dG Monoclonal Antibody (Clone 2E2)

SPECIFICITY: This mouse monoclonal antibody specifically binds to 8-hydroxy-2’- deoxyguanosine within DNA

ISOTYPE: 8-oxo-dG Monoclonal Antibody Staining of a Rat Thymus Section IgG2b.

PREPARATION: This antibody is provided as purified immunoglobulin from mouse ascites in 1X PBS containing 8-oxo-dG MONOCLONAL ANTIBODY 0.01% sodium azide.

172 APPLICATIONS: • ELISA • Immunocytochemistry • Immunohistochemistry

STORAGE: Freeze in working aliquots at -20°C in a manual defrost freezer to avoid repeated freeze-thawings.

ORDERING INFORMATION

4354-MC-050 Anti-8-oxo-dG Monoclonal Antibody (Clone 2E2), 50µl

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OXIDATIVE STRESS

HT Superoxide Dismutase Assay Kit

•- Superoxide Dismutases (SOD) catalyze the dismutation of the superoxide radical O2

into hydrogen peroxide (H2O2) and elemental oxygen (O2), and as such, provides an important defense against the toxicity of superoxide radical (Figure 1). SOD1 and SOD2-deficient mice spontaneously develop liver cancer and are prone to develop tumors, whereas over expression of SOD protects tumor cells from apoptosis. HT SUPEROXIDE DISMUTASE ASSAY

In Trevigen’s HT Superoxide Dismutase Assay, superoxide radical ions, generated from the conversion of xanthine to uric acid, and hydrogen peroxide by xanthine oxidase (XOD), convert WST-1 to WST-1-formazan. WST-1-formazan absorbs light at 450 nm. SODs reduce superoxide ion concentrations and thereby lower the rate of WST-1 formazan formation. The extent of reduction in the appearance of WST-1 formazan is a measure of SOD activity

Figure 1. Hydrogen peroxide production by SODs. present in experimental samples. This assay is free of interference by other catalytic activities, and unlike some other assay kits for SOD, this system is not greatly disturbed by trace metals.

FEATURES: • 96 well format for high throughput screening • Assay performed in as little as 6 minutes • Suitable for mammalian tissues and cell lysates • Suitable for the assay of (Mn2+)-SOD, (Fe2+)-SOD, and (Cu/Zn)-SOD

APPLICATIONS: Calculating relative SOD activity by measuring the percent inhibition of the rate of formation of WST-1 formazan.

COMPONENTS: 173 Catalog # Component Size 7501-500-01 500 Standard, 50 units/µl 50 µl 7501-500-02 10 x SOD Buffer 20 ml 7501-500-03 Xanthine Oxidase (XOD) 3 ml 7501-500-04 10 x Xanthine Solution 2 ml 7501-500-05 20% Triton™ X-100 1 ml 7512-100-06 96-Well Plates 5 7501-500-06 WST-1 Reagent 3 ml

STORAGE: Store components at -20°C, 4°C and room temperature.

REFERENCES: 1. Pouyet, L. and Carrier, A. 2010. Mutant mouse models of oxidative stress. Transgenic Res.

Figure 2. XOD and SOD antagonism in the generation of WST-1 19:155–164. formazan. Xanthine Oxidase (XOD) generates superoxide radical 2. Shanker, M., Willcutts, D., Roth, J.A., Ramesh, R. 2010. Drug resistance in lung cancer. resulting in the reduction of WST-1 by superoxide anion to a colored Lung Cancer: Targets and Therapy 1:23–36. WST-1 formazan product that absorbs light at 450 nm. SOD scavenges 3.Oberley, T.D. 2004. Mitochondria, manganese superoxide dismutase, and cancer. Antioxid. superoxide anion thereby reducing the rate of WST-1 Redox Signal. 6:483-487 formazan generation. 4. Pani, G., R. Colavitti, B. Bedogni, S. Fusco, D. Ferraro, S. Borello, and T. Galeotti. 2004. Mitochondrial superoxide dismutase: a promising target for new anticancer therapies. Curr. Med. Chem. 11:1299-1308.

ORDERING INFORMATION

7501-500-K HT Superoxide Dismutase Assay Kit, 480 Wells

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OXIDATIVE STRESS

Superoxide Dismutase Assay Kit

• - Superoxide Dismutases (SOD) catalyze the dismutation of the superoxide radical (O2 ) into hydrogen

peroxide (H2O2) and elemental oxygen (O2), and as such, provides an important defense against the toxicity of superoxide radical (Figure 1). SOD1 and SOD2-deficient mice spontaneously develop liver cancer and are prone to develop tumors, whereas over expression of SOD protects tumor cells from apoptosis.

In Trevigen’s Superoxide Dismutase Assay, ions generated from the conversion of xanthine to uric acid, and hydrogen peroxide by xanthine oxidase (XOD), convert NBT to NBT-diformazan. NBT-diformazan absorbs light at 550 nm. SODs reduce superoxide ion concentrations and thereby lower the rate of NBT-diformazan formation. The extent of reduction in the appearance of NBT-diformazan is a measure of SOD activity present in experimental samples (Figure 2). The assay is free of interference by other catalytic activities, and is ideal for assaying SOD in mammalian cell lysates. The kit contains the proper lysis buffer and the reagents needed for 100 experimental tests, Figure 1. Hydrogen peroxide production by SODs. 50 positive controls, and 50 negative controls. Unlike some other assay kits for SOD, this system is not greatly disturbed by trace metals. Each assay requires only about five minutes, and after a simple calculation, the percent inhibition of the formation of NBT-diformazan by SOD is converted to the relative activity of the sample.

FEATURES: • Suitable for mammalian cells. • Each sample takes only 5 minutes. • Contains SOD for 50 positive controls. SUPEROXIDE DISMUTASE ASSAY SUPEROXIDE DISMUTASE • Suitable for the assay of (Mn2+)-SOD, (Fe2+)-SOD, and (Cu/Zn)-SOD.

174 APPLICATIONS: Calculating SOD activity by measuring the reduction of NBT-diformazan in cell extracts.

COMPONENTS: Catalog # Component Size 7500-100-01 SOD Standard, 1 unit/µL 200 µl 7500-100-02 25X SOD Reaction Buffer 12 ml 7500-100-03 Xanthine Solution 1.5 ml • 7500-100-04 NBT Solution 6.0 ml 7500-100-05 XOD Solution 2.0 ml 7500-100-06 20X Cell Lysis Solution 12.0 ml

STORAGE: Store components at 4°C.

REFERENCES: Figure 2. XOD and SOD cooperation in the inhibition of 1. Pouyet, L. and Carrier, A. 2010. Mutant mouse models of oxidative stress. Transgenic Res. NBT-diformazan formation. 19:155–164. 2. Shanker, M., Willcutts, D., Roth, J.A., Ramesh, R. 2010. Drug resistance in lung cancer. Lung Cancer: Targets and Therapy 1:23–36.

ORDERING INFORMATION

7500-100-K Superoxide Dismutase Assay Kit, 100 Reactions

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OXIDATIVE STRESS

HT Glutathione Assay Kit

Trevigen’s Glutathione Assay utilizes a carefully optimized enzymatic recycling method, which employs Glutathione Reductase, for the quantification of glutathione. The sulfhydryl group of glutathione reacts with DTNB (5,5’-dithiobis-2-nitrobenzoic acid, Ellman’s reagent) and produces a yellow-colored 5-thio-2-nitrobenzoic acid (TNB). The mixed disulfide, GSTNB (between GSH and TNB) that is concomitantly produced, is reduced by Glutathione Reductase to recycle the GSH and produce more TNB (Figure 1). The rate of TNB production is directly proportional to the concentration of GSH in the sample. Thus, measurement of the absorbance of TNB at 405 or 412 nm provides an accurate estimation of the amount of glutathione in the sample.

FEATURES: • Suitable for mammalian cells, tissue, blood, plasma and other bodily fluids. HT GLUTATHIONE ASSAY • Formatted for 96-well plates. • Contains sufficient reagents to assay 384 data points or to determine GSH in: a. 123 experimental samples, each performed in triplicate, plus one GSH standard curve. b. 108 experimental samples, each performed in triplicate, plus 4 GSH standard curves. c. 88 experimental samples, each performed in triplicate, plus 8 GSH standard curves. • Includes procedure for determining oxidized and reduced glutathione.

APPLICATIONS: Calculating total, reduced, and oxidized glutathione concentrations.

COMPONENTS: Catalog # Component Size 7511-100-01 Glutathione Reductase 80 µl 7511-100-02 25X Assay Buffer 12 ml 7511-100-04 Reaction Mix 8 bottles 175 7511-100-05 96-Well Plates 8 plates 7511-100-06 4 µM GSSG 2.5 ml

STORAGE: Store components at 4°C.

REFERENCES: 1. Tokar, EJ, Qu, W, Liu, J, Liu, W, Webber, MM, Phang, JM, and Waalkes, MP (2010) Arsenic-Specific Stem Cell Selection During Malignant Transformation. JNCI 102:638-49. 2. Coppin JF, Qu W, and Waalkes MP. (2008) Interplay between cellular methyl metabolism and adaptive efflux during oncogenic transformation from chronic arsenic exposure in human cells. J Biol Chem. 283:19342-50. 3. Cabello CM, Bair WB 3rd, Bause AS, and Wondrak GT. (2009) Antimelanoma activity of the redox dye DCPIP (2,6-dichlorophenolindophenol) is antagonized by NQO1. Biochem Pharmacol. 78:344-54. 4. Doudican NA, Bowling B, and Orlow SJ. (2010) Enhancement of arsenic trioxide cytotoxicity by dietary isothiocyanates in human leukemic cells via a reactive oxygen species-dependent mechanism. Leuk Res. 34:229-34.

Glutathione Reductase GSSG + NADPH2 2GSH + NADP

GSH + COO H HOO C SH HOO C SSG HOO C S S + NO 2 Figure 1. Reaction Scheme for O N O 2N 2 O 2N Trevigen’s Glutathione Assay. DTNB TNB GSTNB absorbs at405 nm ORDERING INFORMATION Glutathione HOO C SSG SH Reductase HOO C + GSH

O N 7511-100-K 2 O 2N TNB HT Glutathione Assay Kit, 8 x 96 Wells GSTNB

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OXIDATIVE STRESS

HT Glutathione Peroxidase Assay Kit

Glutathione peroxidase (GP) is found in mammalian cells and helps to prevent lipid peroxidation of cell membranes by consuming free peroxide in the cell. Thus, Glutathione Peroxidase plays a critical role in protecting the cell from free radical damage. Tetrameric Glutathione Peroxidase

catalyzes the reduction of H2O2 to water, and organic peroxides to the corresponding stable alcohols, by using glutathione as a source of reducing equivalents. It requires selenium as a cofactor and contains a selenocysteine amino acid residue in the active site of each monomer that participates in the actual mechanism of the enzyme. Glutathione Reductase (GR), provided with each kit, then reduces the oxidized glutathione to complete the cycle (Figure 1). The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm. The rate of decrease in the absorbance at 340 nm is directly proportional to the Glutathione Peroxidase activity in the sample. Trevigen’s Glutathione Peroxidase Assay Kit can be used to measure glutathione-dependent peroxidases in plasma, erythrocyte lysates, tissue homogenates, and cell lysates. GP catalyzes the following reaction: FEATURES: • Suitable for a 96 well or cuvette format • GP Suitable for plasma, erythrocyte lysates, tissue homogenates, and cell lysates 2GSH + H2O2 GSSG + 2H2O APPLICATIONS: Calculating glutathione peroxidase activity

COMPONENTS: GR + + Catalog # Component Size

HT GLUTATHIONE PEROXIDASE ASSAY HT GLUTATHIONE GSSG + NADPH + H 2GSH + NADP 7512-100-01 Glutathione Peroxidase 800 µl 7512-100-02 10X Assay Buffer 20 ml 176 7512-100-03 GSH+NADPH 10 vials 7512-100-04 Glutathione Reductase 1.1 ml Glutathione Reductase (GR) then reduces the oxidized glutathione to 7512-100-05 Cumene Hydroperoxide 12 ml complete the cycle. 7512-100-06 96-well plates 5 plates

STORAGE: Components are stored at -20°C and room temperature.

REFERENCES: 1. Ozdemir G, Ozden M, Maral H, Kuskay S, Cetinalp P, Tarkun I. Malondialdehyde, glutathione, glutathione peroxidase and homocysteine levels in type 2 diabetic patients with and without microalbuminuria. Ann Clin Biochem. 2005, 42:99-104. 2. Prasad NR, Menon VP, Vasudev V, Pugalendi KV. Radioprotective effect of sesamol on gamma-radiation induced DNA damage, lipid peroxidation and antioxidants levels in cultured human lymphocytes. Toxicology. 2005, 209:225-35. 3. Sindhu RK, Ehdaie A, Farmand F, Dhaliwal KK, Nguyen T, Zhan CD, Roberts CK, Vaziri ND. Expression of catalase and glutathione peroxidase in renal insufficiency. Biochim Biophys Acta. 2005, 1743:86-92. 4. Mukhopadhyay P, Rajesh M, Bátkai S, Kashiwaya Y, Haskó G, Liaudet L, Szabó C, Pacher P. Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. Am J Physiol Heart Circ Physiol. 2009 296:H1466-83.

STORAGE: Store components at -20˚C, and 4˚C.

REFERENCES: 1. Chu, F.F., R.S. Esworthy, J.H. Doroshow. 2004. Role of Se-dependent glutathione peroxidases in gastrointestinal inflammation and cancer. Free Radic. Biol. Med. 36(12):1481-1495.

ORDERING INFORMATION

7512-100-K HT Glutathione Peroxidase Assay Kit, 5 x 96 Wells

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OXIDATIVE STRESS

HT Glutathione Reductase Assay Kit

This high throughput glutathione reductase assay kit is designed to measure cellular levels of Glutathione Reductase, an enzyme which catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) (Figure 1). GSH is an important intracellular antioxidant that reacts with oxygen free radicals and organic peroxides and acts as a substrate for other detoxification enzymes such as glutathione peroxidase and glutathione S-transferase. The activity of glutathione reductase is an important measure of the antioxidant status of the cell. Glutathione HT GLUTATHIONE REDUCTASE ASSAY reductase requires NADPH for its activity, resulting in the reduction of GSSG to GSH and the corresponding oxidation of NADPH to NADP+.

Trevigen's Glutathione Reductase Assay Kit is a spectrophotometric assay in which the oxidation of NADPH to NADP+ is monitored by the decrease in absorbance at 340 nm. This rate of decrease in absorbance at 340 nm is directly proportional to the glutathione reductase activity in the sample because the enzyme is present at rate limiting concentrations. This kit provides the user with all the reagents and plates to easily and rapidly assay for glutathione reductase in cell and tissue extracts.

FEATURES: • Suitable for cell lysates, erythrocyte lysates, and tissue homogenate • Sufficient reagents for 500 assays • 96 well format for high throughput screening

APPLICATIONS: Calculating glutathione reductase activity.

COMPONENTS: Catalog # Component Size 177 7510-100-03 NADPH 10 x 9.2mg 7512-100-06 96-well plates 5 plates 7513-500-01 Glutathione Reducatase 1 ml 7513-500-02 10X GR Buffer 20 ml 7513-500-03 GSSG 3 ml 7501-500-05 20% Triton X -100 1 ml

STORAGE: Store components at -20°C, 4°C, and room temperature.

REFERENCES: 1. Berg JM, Tymoczko JL, Styer L. 2002. Biochemistry, 5th Edition, WH Freeman and Company, New York. 2. Tietze, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem., 27, 502-522 (1969). 3. Smith IK, Vierheller TL, Thorne CA. Assay of glutathione reductase in crude tissue homogenates using 5,5'-dithiobis(2-nitrobenzoic acid). Anal. Biochem., 175, 408-413 (1988). 4. Dolphin, D, Poulson, R, Avramovic, O. (Eds.), Glutathione: Chemical, Biochemical, and Metabolic Aspects, Volumes A and B, Wiley and Sons (1989). 5. Dringen R, Gutterer JM. Glutathione reductase from bovine brain. Methods Enzymol. 348, 281-288 (2002).

GLUTATHIONE REDUCTASE GSSG + NADPH + H+ 2GSH + NADP+ ORDERING INFORMATION (Absorbs at 340 nm) 7513-500-K HT Glutathione Reductase Assay Kit, 5 x 96 Wells Figure 1. Reduction of glutathione disulfide (GSSG) by glutathione reductase and NADPH

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OXIDATIVE STRESS

Glutathione Reductase Assay Kit

Trevigen’s Glutathione Reductase Assay Kit is a higher volume assay designed to measure cellular levels of Glutathione Reductase, an enzyme which catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) (figure1). GSH is an important intracellular antioxidant that reacts with oxygen free radicals and organic peroxides and acts as a substrate for other detoxification enzymes such as glutathione peroxidase and glutathione S-transferase. The activity of glutathione reductase is an important measure of the antioxidant status of the cell. Glutathione reductase requires NADPH for its activity, resulting in the reduction of GSSG to GSH and the corresponding oxidation of NADPH to NADP+.

Trevigen’s Glutathione Reductase Assay takes less than 6 minutes to perform and is based on the measurement of the loss in absorbance at 340 nm due to the conversion of NADPH to NADP+. The kit contains everything that you need to perform the assay on 100 samples and its corresponding blanks, each in duplicate, as well as 10 standard curves with each point performed in duplicate. In total, each kit can generate data on over 500 data points. The NADPH is provided lyophilized in 10 vials, each containing sufficient NADPH for generating one standard curve and for determining glutathione reductase levels in 10 experimental samples.

FEATURES: • Suitable for cell lysates, erythrocyte lysates, and tissue homogenates • Each sample takes only 6 minutes • Sufficient reagents for 100 assays and 10 standard curves GLUTATHIONE REDUCTASE ASSAY REDUCTASE GLUTATHIONE APPLICATIONS: Determination of glutathione reductase activity.

178 COMPONENTS: Catalog # Component Size 7510-100-01 Glutathione Reductase 500 µl 7510-100-02 25X Glutathione Reductase 20 ml Reaction Buffer 7510-100-03 NADPH 10 x 9.2mg 7510-100-04 GSSG Solution 6 ml 7510-100-05 25X Sample Dilution Buffer 20 ml 7510-100-06 25X Tissue Homogenization 40 ml Buffer

STORAGE: Store components at 4°C. NADPH should be stored at -20°C.

REFERENCES: 1. Nordman, T., L. Xia, L. Bjorkhem-Bergman, A. Damdimopoulos, I. Nalvarte, E.S. Arner, G. Spyrou, L.C. Eriksson, M. Bjornstedt, and J.M. Olsson. 2003. Regeneration of the antioxidant ubiquinol by lipoamide dehydrogenase, thioredoxin reductase and glutathione reductase. Biofactors 18:45-50. 2. Droge, W. 2003. Oxidative stress and aging. Adv. Exp. Med. Biol. 543:191-200. 3. Higuchi, Y. 2003. Chromosomal DNA fragmentation in apoptosis and necrosis induced by oxidative stress. Biochem. Pharmacol. 15:1527-1535.

Glutathione Reductase

2 HS

ORDERING INFORMATION

++ NADPH + H NADP

7510-100-K

Glutathione Reductase Assay Kit,

500 Data Points, 100 Assays

1-800-873-8443 • www.trevigen.com Figure 1. Reduction of glutathione disulfide (GSSG) by glutathione reductase and NADPH. INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:25 PM Page 183 MOLECULAR BIOLOGY REAGENTS

DNA Damage and Genomic Instability

Cancer Cell Behavior

Apoptosis Detection

Stem Cell Products

Oxidative Stress

MOLECULAR BIOLOGY REAGENTS MOLECULAR BIOLOGY REAGENTS MOLECULAR BIOLOGY INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:25 PM Page 184

MOLECULAR BIOLOGY REAGENTS

Overview

Trevigen’s Molecular Biology products encompass Nucleic Acid extraction, DNA electrophoresis and staining, and Molecular Biology reagents and enzymes. This collection reflects Trevigen’s commitment to high quality research reagents and our tradition of developing novel products. All of these specialized products were developed for use in our own labs, and are routinely used by our research and development group, thereby assuring the highest quality and best technical support.

Trevigen’s Genomic DNA Isolation Kit is a non-phenol:chloroform-based, biphasic extraction that overcomes the common problems of endogenous nucleases and shearing associated with genomic DNA isolation. No columns or multiple organic extractions are required. DNA isolated with these kits is suitable for PCR, DNA hybridization, array-based experiments and restriction enzyme digestions.

TreviGel™ is a DNA electrophoresis matrix that provides high resolution separation of DNA fragments from 50 to 1500 bp (TreviGel™ 500) or 200 to 25,000 bp (TreviGel™ 5000). TreviGel™ is stronger, clearer, and provides better resolution than most agaroses and agarose blends. TreviGel™ 500 has a high melting temperature allowing you to perform high speed (high voltage) electrophoresis without sacrificing the quality of your results. The Electrophoresis accessories, Orange G loading dye and TAE buffer are also available.

Trevigen offers high purity proteins and enzymes for DNA labeling, including the terminal deoxynucleotidyl transferase (TdT). Three types of sheared DNA are available for hybridization MOLECULAR BIOLOGY REAGENTS OVERVIEW solutions. We also have proteinase K for permeabilizing cells and tissues. 180

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MOLECULAR BIOLOGY REAGENTS

Genomic DNA Isolation Kit

Trevigen’s Genomic DNA Isolation kit is designed to overcome the most common problems associated with genomic DNA isolation, namely endogenous nucleases and shearing. The kit employs a non-phenol:chloroform-based biphasic extraction to prepare highly purified genomic DNA preparations, free from protein and RNA. DNA isolated using the kit is suitable for PCR, DNA hybridization, array-based experiments and restriction enzyme digestion. No columns or multiple organic extractions are required.

FEATURES: • Simple biphasic extraction • Phenol and chloroform free • Protects DNA from endogenous nucleases • No columns or special equipment required

APPLICATIONS: Isolation of genomic DNA DNA ISOLATION COMPONENTS: Catalog # 4850-20-GD: For the isolation of DNA from Cultured Cells

Catalog # Component Size 4850-20-01 Lysis Solution 1 2 x 1 ml 4850-20-02 Extraction Solution 2 20 ml 4850-20-03 Extraction Buffer 3 8 ml 4850-20-04 Sodium Acetate 4 1 ml 4850-20-05 DNase-free Water 5 2 ml 4850-20-06 Sample Buffer 2 ml 181

Catalog # 4859-20-K: Additional Buffers required for the isolation of DNA from tissues

Catalog # Component Size 4850-20-06 Sample Buffer 2 ml 4859-20-01 10X Tissue Buffer 500 µl

RELATED PRODUCTS: Orange G Buffer TAE Buffer TreviGel™ 5000

STORAGE: Store kit at room temperature.

ORDERING INFORMATION

4850-20-GD Genomic DNA Isolation Kit, 20 Samples 4859-20-K Tissue Extraction Reagents, 20 Samples

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APOPTOSISMOLECULAR DETECTION BIOLOGY REAGENTS

TreviGel™ 500 and 5000

An alternative to agarose, TreviGel™ is a unique blend of polysaccharides containing AgaCryl™ Apoptosisproducing waselectrophoretic originally defined gels with by unsurpassedthe morphological resolving changes capabilities, that occur strength, with remarkableand clarity. fidelityTwo formulations in a wide variety tailored of for cell PCR types, fragment regardless separations of cell lineageare available. and the method of cell death induction. The morphological changes can occur rapidly after induction and include cell surface blebbing,TreviGel™ and 500 cell is ideal and fornuclear separating shrinkage. small The DNA morphological fragments generated changes by are PCR. best TreviGel™ described 500 in thymocytesis available asin vitroa powder, and Figurein several 1 provides convenient an illustration sizes. The of concentration typical cell morphologies of the powder that can may be beadjusted observed to allow during the a 24optimal hour separationperiod after of induction DNA fragments of apoptosis. from 50Consideration to 1500 bp. of The morphology powder is isquickly the single and mosteasily important prepared methodby heating for detectionin TAE buffer of apoptosis that is typicallyand no other used biochemical for agarose test gel orelectrophoresis. assay is available This to product replace approaches microscopic polyacrylamide observation. in its separation properties, but does not have the neurotoxic properties of acrylamide.

TreviGel™ 5000 is optimized for the separation of DNA fragments in the range of 200 bp to Researchers25,000 bp. TreviGel™ require alternative5000 powder methods is available for scoring in several apoptosis sizes and that includes provide easy supportive to follow biochemical evidence or quantitation of data. This can be particularly important in vivo when 1.5% TreviGel™ 500. Lane 1) 2 µg of F174/Hae III and Lane instructions for use. The powder is quickly and easily prepared by heating in TAE buffer. This 2) 2 µg of a 100 bp ladder. The size markers to the side theproduct later replacesmore obvious agarose morphological in its separation changes properties. may not With occur a lower due cost to phagocytosis per gel than agarose,and cell of each lane indicate the number of base pairs. Gels were removal,TreviGel™ and 5000 in achievesexperimental better systems separation, when and large has thesample advantages numbers of beinghave toclearer be assayed (enhancing and prepared with 1X TAE buffer and DNA was visualized by screened.sensitivity) Once and stronger.morphological evidence has determined that the mechanism of cell death ethidium bromide staining. occurs by apoptosis, continued stringent analysis using morphology is not necessary and a moreFEATURES: convenient biochemical method is more appropriate. As elucidation of the complex and varied• Strong: biochemical High gel strengthpathways helps leading prevent to the sample execution loss of apoptosis continues, it is likely that the• Clear: options DNA available fragments for apoptosisreally stand detection out will continue to expand. • Cost effective: Use less than other speciality agaroses Please check our website for the latest product additions and product information. OVERVIEW ELECTROPHORESIS PRODUCTS • Fast: High voltage capable

18200 APPLICATIONS: • Gel electrophoresis Cell• Mobility Membrane shift assays Events • Download our TreviGel™ Product Application Guide from our website.

PRODUCTSTREVIGEL™ OFFERED: 500 SEPARATION SIZE TABLE ANNEXINGel Concentration V CONJUGATES Fragment Sizes (bp) IMAGE MORPHOLOGY 0.5 300 - 1500 Some 1.0 of the earliest apoptotic200 - 1000 changes occur at the cell surface. The early recognition of apoptotic cells 1.5 by phagocytic cells,150 the - significant 800 loss of water leading to cell shrinkage, and the maintenance of 2.0intact cell membranes,50 despite- 600 the cell surface blebbing observed in many cell types, indicates TreviGel™ 5000. Lane 1) 250 ng of a 100 bp ladder and Lane 2) 250 ng of a 1 kb ladder. The size markers to the significant changes have occurred in the plasma membrane. One of the better understood cell surface TREVIGEL™ 5000 SEPARATION SIZE TABLE sideFigure of each 1: laneA schematic indicate therepresentation number of baseof cells pairs. undergoing Gels modifications is exposure of phosphatidylserine (PS). Normally, PS is confined to the inner layer of wereapoptosis prepared (clockwise with 1X TAE from buffer top) and or DNAnecrosis was (counterclock-visualized theGel lipid Concentration bilayer. This asymmetric Fragment Sizedistribution (kbp) is maintained in normal cells by the action of specific by ethidiumwise from bromide top). Apoptoticstaining. cells undergo a series of mor- proteins. 0.5 After induction2 - of25 apoptosis, under the influence of translocases, the PS is flipped from the 0.6 inner to the outer 1bilayer - 20 rendering the molecule available for detection. Annexin V is a blood clotting 0.7 factor that exhibits0.8 - 10a high calcium-dependent specificity for PS binding. The coupling of ORDERING INFORMATION annexin 1.0 to fluorescein,0.5 or -biotin, 7 generates a direct, rapid, and simple method for the detection of 1.2 0.4 - 6 9804-050-P apoptosis on unfixed cells. Figure 2 is a depiction of PS flipping during apoptosis and subsequent TreviGel™ 500, 50 g detection 1.5 using Annexin0.2 V. - 3 2.0 0.1 - 2 9804-100-P TreviGel™ 500, 100 g STORAGE: Store at room temperature. 9804-250-P TreviGel™ 500, 250 g REFERENCES: 9806-050-P 1. Vanek, P.G., S.J. Fabian, C.L. Fisher, J.G. Chirikjian, and G.B. Collier. 1995. Alternative to TreviGel™ 5000, 50 g polyacrylamide gels improves the electrophoretic mobility shift assay. BioTechniques 18:704-706. 9806-100-P 2. Fairbrother, W.G. and L.A. Chasin. 2000. Human genomic sequences that inhibit splicing. TreviGel™ 5000, 100 g Molecular and Cellular Biology 20(18): 6816-6825. 9806-250-P TreviGel™ 5000, 250 g Bulk quantities available. Please inquire.

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APOPTOSISMOLECULAR DETECTION BIOLOGY REAGENTS

Orange G Loading Dye

The Orange G Loading Dye is ideal for PCR applications. Unlike methylene blue containing PRODUCTSloading dyes, OFFERED: the Orange G dye migrates with DNA between 10 and 20 nucleotides long. ANTI-CLEAVEDThe migration at this CASPASE-3, size prevents theANTI-PARP, obscuring of PARP larger DNAAPOPTOSIS bands, and ASSAYconveniently KITS,indicates BCL-2 the front FAMILY of DNA migration.ANTIBODIES, This Ficoll-based DEPSIPHER™, dye is provided MITOSHIFT™ at a 5X concentration and does not require refrigeration. Although organelles remain grossly normal during the earlier stages of apoptosis, recent research IMAGE DEPSIPHER.TIF hasREFERENCE: revealed a pivotal role for mitochondria in the apoptotic cascade. The energy generated during cellular1. Siles, respiration B.A., G.B. accumulates Collier, D.J. in Reeder,the mitochondrial and W.E. May.transmembrane 1996. The spaceuse of as a annew electron gel matrix gradient for calledthe separation the mitochondrial of DNA fragments:membrane apotential comparison or Δψ studym. Thisbetween electrochemical slab gel electrophoresis gradient is often and ELECTROPHORESIS PRODUCTS disturbedcapillary during electrophoresis. apoptosis and Appl. can Theor. be detected Electrophor. using cationic 6:15-22. dyes such as DePsipher™ (5,5’6,6’- tetrachloro-1,1’,3,3’-tetraethylbenz-imidazolylcarbocyanine iodide) or MitoShift™ (tetramethylrhodamine ethyl ester). DePsipher™ readily enters cells and accumulates as a multimeric aggregate Figure 3: Identification of apoptotic cells using DePsipher™. within50X healthy TAE mitochondria, Electrophoresis and under UV light,Buffer this multimeric form emits red light. In apoptotic INT407 human cells were treated with 25 µM etoposide for 6 hours, and then incubated with the DePsipher™ reagent cells, the mitochondrial membrane potential collapses and the dye returns to a monomeric form. in Reaction Buffer for 30 minutes prior to visualization. TheTrevigen’s monomeric TAE molecule Buffer is emitsprepared green to exactingfluorescence specifications under UV light.ensuring DePsipher™ consistent provides electrophoresis a rapid Healthy cells (containing red aggregates) can be differen- results from run to run. The buffer is thoroughly qualified using our TreviGel™ products to

fluorescence-based assay for the apoptosis-associated loss of mitochondrial potential (Figure 3). OVERVIEW tiated from apoptotic cells (containing mostly green assure the highest possible resolution. The buffer is provided in 500 ml polypropylene, monomers) Activationshatter-proof of caspases, bottles. or cysteinyl proteases, is a necessary event for execution of the apoptotic response. Some of the caspases are activated early in the apoptotic process and their activation is the first step in a cascade of proteolytic cleavage of key proteins and enzymes, including other caspases25X PBS and poly (ADP-ribose) polymerase (PARP). The caspases each cleave a defined amino acid sequence and this specificity has led to the development of highly specific irreversible peptide inhibitors.Trevigen Deliverynow offers of these an economical peptides allows 25X forPBS complete solution inhibition for your of research the execution needs. of Scientists apoptosis, at therebyTrevigen providing routinely a means use our to 25Xprobe PBS the inearly ELISA, events immunocytochemistry, in apoptosis and to investigate and in Western the ordering Blots. ofTrevigen’s key events 25X in the PBS process. is supplied Since inthe 500 substrate ml plastic specificity bottles of and the containscaspases 250is high, mM analysis Potassium of substratePhosphate cleavage (pH 7.4), also 3.625Mprovides NaCl.a useful A 1Xbiochemical solution containsmarker. Figure 10 mM 4 showsPotassium a Western Phosphate blot 18300 detecting(pH 7.4), cleaved 145 mM PARP NaCl. in apoptotic HL-60 cells compared to a population of mostly healthy cells. Figure 5 shows the presence of cleaved caspase-3 in Jurkat cells after treatment to induce apoptosis.

Western blot analysis of Wehi, Jurkat and CCL-119 cell The Bcl-2 family of proteins is well known to be important in determining cell fate although their lines untreated (H) and treated (T) with 25 µM Etopo-side mechanism of action remains unclear. The relative levels of the family members as well as the for 16 hours at 37oC. Cells were lysed in Tris-Glycine SDS sample buffer at the concentration 1 x 107 cells/ml and 10 subcellular distribution of these proteins changes during apoptosis. In particular, the movement µl of each lysate were loaded per well of a 4-20% Tris- of some members from the cytoplasm to the mitochondria and the subsequent associations that Glycine gel. Proteins were transferred onto an Immobilon occur between the Bcl-2 family members and other mitochondrial membrane associated proteins FL membrane and PARP was detected with Trevigen’s anti- are indicated to be crucial steps in apoptosis. Antibodies that recognize the individual Bcl-2 family PARP (C2-10) antibody followed by an IR800-conjugated secondary antibody. The membrane was scanned using an members provide powerful tools for studying alterations in expression levels, the subcellular Odyssey Infrared Imaging System (Licor). The 116 kDa redistribution, and protein associations of these intriguing molecules. band corresponds to full length PARP and the 85 kDa band is the apoptotic cleavage product.

ORDERING INFORMATION

9850-250-6 5X Orange G Loading Dye, 6 x 250 µl 9860-500-2 50X TAE Electrophoresis Buffer, 2 x 500 ml 9860-500 50X TAE Electrophoresis Buffer, 500 ml 4868-500-06 25X PBS, 6 x 500 ml

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MOLECULAR BIOLOGY REAGENTS

Terminal Deoxynucleotidyl Transferase (TdT)

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase that catalyzes the template-independent addition of deoxyribonucleotides to the 3’ hydroxyl terminus of single- or double-stranded DNA. Trevigen’s TdT is highly purified and qualified for in situ labeling.

CONCENTRATION:15 units/µl.

UNIT DEFINITION: 1 unit incorporates 1 nmole of dATP into acid-insoluble form in 1 hour at 37˚C using activated DNA as a template/primer.

APPLICATIONS: • In situ labeling of apoptotic cells • End labeling of oligonucleotides

SOURCE: Purified from a recombinant source.

STORAGE: Store at -20˚C in a manual defrost freezer to avoid repeated freeze-thawing. Calf Thymus DNA, Herring Sperm DNA, Salmon Sperm DNA

Highly purified and qualified for Northern, Southern, probe array, and dot blotting procedures, Trevigen’s sheared DNA preparations are provided at 10 mg/ml with a fragment size range of 80–500 bp. The format allows convenient addition to buffers to create a final recommended MOLECULAR BIOLOGY REAGENTS working concentration of 0.1 mg/ml. Quality control testing includes concentration determination, 184 fragment sizing by gel electrophoresis, A260/A280 ultraviolet absorbance ratio, and protein content. Proteinase K

Proteinase K is used for permeabilizing cells and tissues. It is used in our TACS® In Situ Apoptosis Detection kits prior to labeling. Proteinase K can also be used to remove contaminating/unwanted nucleases or other proteins.

ORDERING INFORMATION

9510-01K-EB Terminal Deoxynucleotidyl Transferase Enzyme, 1000 Units 9600-5-D Calf Thymus DNA, 5 x 1 ml 9605-5-D Herring Sperm DNA, 5 x 1 ml 9610-5-D Salmon Sperm DNA, 5 x 1 ml 4800-30-01 Proteinase K, 30 mg in 30 µl

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COMETASSAY®

PARP ASSAY

PARG ASSAY

PHARMACODYNAMIC PARP ASSAY

CELL INVASION ASSAY

TUBE FORMATION ASSAY TREVIGEN CELL ASSAYS (TCA) SERVICES (TCA) TREVIGEN CELL ASSAYS INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:26 PM Page 190

TREVIGEN CELL ASSAYS SERVICES

Trevigen Cell Assays (TCA)

Trevigen Cell Assays (TCA), a division of Trevigen, Inc., was established in 2008 to conduct contract research work for the pharmaceutical, biotechnology, government and academic segments of the medical research market. TCA specializes in designing and conducting assays for lead compounds and genotoxic screening based on DNA damage and repair as well as cancer cell behavior.

PROCESS: • A Project Manager and a Senior Scientist is assigned for every project • A detailed Protocol and Schedule is prepared before the work begins • Site training is available if applicable • Observations, comments, and updates are sent by e-mail during the study • A Final Report with Statistical/Graphical analysis is provided

ADVANTAGES: • The technical team at TCA developed the assays and is unmatched in skill and knowledge • Confidentiality is assured. A Non-Disclosure Agreement can be executed if requested

TCA Service: Assay: Applications:

Rapid analysis of DNA • Detect and quantitate DNA danage CometAssay® fragmentation associated with • Follow DNA repair

TREVIGEN CELL ASSAYS SERVICES TREVIGEN CELL ASSAYS DNA damage. Measurement of the biotinylated • Assay inhibitors and activators of PARP activity PARP Assay poly(ADP-ribose) incorporation 186 • Determination of IC values for PARP inhibitors onto histone proteins. 50 • Identify inhibitors and activators of Measurement of the loss of PARG Assay PARG activity biotinylated PAR from histones. • Determination of IC50 values for PARG inhibitors

• Quantification of PAR in peripheral blood mononuclear cells, tissue culture cells, and tumor lysates from different tissues, organs Pharmacodynamic Measurement of net PAR levels and xenografts PARP Assay in cellular extracts and tumor • Monitoring the efficacy of PARP inhibitors on PAR lysates. formation in vivo • Facilitating development of PARP and PARG targeted therapeutics

Screening for compounds that influence cellular invasion through Analysis of responses to • Extracellular matrices Cell Invasion Assay chemokines, toxins, drugs and • Angiogenesis other analytes of interest. • Immune responses • Tumor cell metastasis

Tube Formation Investigation of prospective • Detection of inducers and inhibitors Assay angiogenesis inhibitors. of endothelial cell tube formation

Please complete and submit Custom Quotation Form, which you can find online at www.trevigen.com by clicking on the SERVICES tab. For more information contact us at [email protected] or 1-800-873-8443.

1-800-873-8443 • www.trevigen.com OVERVIEWPRICE LIST 00 187

95 95 g)/VEGF(600 ng) 60 µl 95 µ Angiogenesis Angiogenesis 24 Well BME24 Well BME 96 Well Laminin I 24 Well Laminin I 96 Well Collagen I24 Well Collagen I 96 Well Poly-L-Lysine Poly-L-Lysine 100 ml Poly-D-Lysine 100 ml Collagen I (Rat) 1 ml Collagen I (Rat) 100 mg 91 Collagen I (Bovine) 1 ml 90 Collagen I (Bovine) 50 mg 85 High Protein Concentration 85 87 87 In Vivo In Vivo ® ® ® ® ® ® ® ® ® ® ® ® ® Cell Invasion Assay 96 samples 100 Cell Invasion Assay 24 tests 100 Cell Invasion Assay 96 samples Cell Invasion Assay 24 tests 100 Cell Invasion Assay 96 samples 100 Cell Invasion Assay 24 tests 100 100 Cell Proliferation Assay-Core Kit 96 Tests Cell Proliferation Assay-Core Kit 96 Tests Cell Proliferation Assay Kit 96 Tests 108 Cell Proliferation Assay Kit 96 Tests 108 Cell Proliferation Assay Kit 96 Tests 108 (3448-020-01 & 3448-020-02) 100 ml each 108 (3448-020-03 & 3448-020-04) 10 ml / 1 ml 109 109 (3450-048-01 & 3450-048-02) 48 reactors Assay Inhibition Kit 48 samples 95 Activation Kit 48 samples 95 (FGF/VEGF & AngioRack included) 48 tests 95 95 BME (PC) 5 ml 74 3444-005-02 Cultrex 3456-024-K Cultrex 3457-024-K Cultrex 3448-020-CH Harvest 3-D Culture Cell Buffers 3448-020-CL 3-D Culture Cell Loading Buffers 3450-048-SK Starter Kit DIVAA 3455-024-K Cultrex 3445-096-K 3-D Culture BME 3450-048-K Directed 3445-096-CK 3-D Culture 3455-096-01 Cell Invasion Chamber each 3455-096-02 5X BME Solution 1 ml 3455-096-03 10X Coating Solution 1 ml 3455-096-04 Buffer 2 x 1.5 ml 25X Cell Wash 100 3455-096-05 10X Cell Dissociation Solution 2 x 1.5 ml 100 3455-096-K Cultrex 100 100 100 3456-096-02 5X Laminin I Solution 1 ml 3456-096-K Cultrex 100 3457-096-02 5X Collagen I Solution 1 ml 3457-096-K Cultrex 100 3445-096-02 3-D Culture Cell Proliferation Reagent 3 ml 3445-096-03 Strip Plates 3-D Culture 96 Well 2 plates 108 108 3446-005-01 3-D Culture Laminin I 30 mg 3446-096-K I 3-D Culture Laminin 92, 106 3447-020-01 100 mg 3-D Collagen I Rat Tail 3447-096-K I 3-D Culture Collagen 93, 107 3448-020-K 3D Culture Cell Harvesting Kit 20 tests 3450-048-03 Buffer Wash DIVAA 25 mL 3450-048-04 FGF-2 Positive Control DIVAA 100 ng/10 uL 3450-048-05 Cell Sperse DIVAA 15 mL 109 3450-048-08 Heparin Solution 10 µl: 2mg/ml 95 3450-048-09 AngioRack 48 positions 95 3450-048-B10 FGF-2 (1.8 DIVAA 95 3450-048-B9 FGF-2 (300 ng)/VEGF(100 ng) 10 µl DIVAA Angioreactor with BME 3450-048-DA DIVAA 95 3450-048-FL 200X FITC Lectin & Diluent 50 µl & 400 µl 95 3450-048-IK Directed CATALOG # DESCRIPTIONCATALOG SIZE LIST PRICE PAGE 3438-100-01 Cultrex 3439-100-01 Cultrex 3440-005-01 Cultrex 3440-100-01 Cultrex 3442-005-01 Cultrex 3442-050-01 Cultrex 3445-001-01 3-D Culture Matrix ml 1 3445-005-01 3-D Culture Matrix ml 5 105 76, 105 76,

75,165 Trevigen Price List Trevigen PRICE LIST PRICE BME, no phenol red, BME, no phenol red, BME, no phenol red, reduced BME, no phenol red, reduced CS Solution 100 ml Solution CS Wash 100 ml Cell Staining Kit 100 ml Each 104 104 104 BME with phenol red, BME with phenol red, RGF BME with phenol red, RGF BME with phenol red, RGF BME, no phenol red 1 ml BME, no phenol red (PC) 1 ml BME, no phenol red 5 ml BME, no phenol red (PC) 5 ml 70 BME, no phenol red, 72 70 BME, no phenol red, 72 Human BME PathClear 1 mg/ 1ml Fibronectin 1 mg Vitronectin 50 µg Human Fibronectin, PathClear 1 mg 80 Human Vitronectin, PathClear 50 µg 88 Human Vitronectin, 50 µg 89 81BME with phenol red 82 BME with phenol red BME with phenol red BME with phenol red Laminin (mouse) 1 mg Laminin (mouse) (PC) 1 mg Collagen IV 1 mg 83 84 86 www.trevigen.com ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® • reduced growth factor reduced growth factor 5 ml reduced growth factor (PC) 5 ml Stem Cell Qualified (PC) 70 growth factor, 1 ml 72 Stem Cell Qualified (PC) growth factor, 5 ml 75 (3433-001-01 & 3430-50-01) 1 ml (PC) (3433-001-02 & 3430-50-01) 1 ml 70 (3433-005-01 & 3430-50-01) 5 ml (PC) (3433-005-02 & 3430-50-01) 72 5 ml 70 72 reduced growth factor 1 ml reduced growth factor (PC) 1 ml 70 72 Nucleic Acids Reduced, PathClear 1ml @ 1 mg/ (3432-001-01 & 3430-50-01) 1 ml (PC) (3432-001-02 & 3430-50-01) 82 1 ml 70 (3432-005-01 & 3430-50-01) 5 ml (PC) (3432-005-02 & 3430-50-01) 72 5 ml 70 72 (Clone YTH-5B7) (Clone YTH-5B7) 100 µg (Clone YTH-6A7) 100 µg 149 (Clone YTH-2D2) 100 µg 149 (Clone YTH-10C4) 100 µg (Clone YTH-8C8) 149 100 µg (Clone YTH-2H12) 151 100 µg 151 152 3437-100-K Cultrex 3433-001-01 Cultrex 3422-001-01 Cultrex 3433-005-02 Cultrex 3434-001-02 Cultrex 3434-005-02 Cultrex 3431-001-02 Cultrex 3431-005-01 Cultrex 3431-005-02 Cultrex 3433-001-02 Cultrex 3433-005-01 Cultrex 3430-001-01 Cultrex 3430-001-02 Cultrex 3430-005-01 Cultrex 3430-005-02 Cultrex 2281-MC-100 Anti-Bax Monoclonal Antibody 2282-MC-100 Anti-Bax Monoclonal Antibody 2290-MC-100 Anti-Bcl-2 Monoclonal Antibody 2291-MC-100 Anti-Bcl-2 Monoclonal Antibody 2300-MC-100 Anti-Bcl-XL Monoclonal Antibody 3437-100-01 Cultrex 3437-100-02 Cultrex 3432-001-01 Cultrex 3432-001-02 Cultrex 3432-005-01 Cultrex 3432-005-02 Cultrex 3415-001-02 Cultrex 3416-001-01 Cultrex 3417-001-01 Cultrex 3420-001-01 Cultrex 3421-001-01 Cultrex 3430-50-06 Phenol Red Solution 6 x 50 µl 3431-001-01 Cultrex 70 2305-PC-020 Anti-Cleaved-Caspase-3 PC Sample 20 µl 2305-PC-100 Anti-Cleaved-Caspase-3 PC 100 µl 3400-010-01 Cultrex 3400-010-02 Cultrex 154 3410-010-01 Cultrex 154 CATALOG # DESCRIPTIONCATALOG SIZE LIST PRICE PAGE Antibody 2275-PC-020 Human Anti-G3PDH 20 µl Antibody 2275-PC-100 Human Anti-G3PDH 100 µl 2280-MC-100 Anti-Bax Monoclonal Antibody 155 155 1-800-873-8443 INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:26 PM Page 191 Page PM 2:26 1 1/31/11 INT_Trevigen_Catalog_2011_1-31:Layout INT_Trevigen_Catalog_2011_1-31:Layout 1 1/31/11 2:26 PM Page 192

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CATALOG # DESCRIPTION SIZE LIST PRICE PAGE CATALOG # DESCRIPTION SIZE LIST PRICE PAGE 3458-024-K Cultrex® 24 Well Collagen IV 3850-100-01 8-oxo-dG Oligonucleotide 100 pmol 44 Cell Invasion Assay 24 tests 100 3850-100-OL 8-oxo-dG Oligo and Complement A 100 pmol each 44 3458-096-02 5X Collagen IV Solution 1 ml 100 3900-200-08 10X REC Buffer 8 1 ml 46 3458-096-K Cultrex® 96 Well Collagen IV 3900-500-01 10X REC Buffer 1 1 ml 56 Cell Invasion Assay 96 samples 100 3900-500-04 10X REC Buffer 4 1 ml 49, 62, 63 3465-024-K Cultrex® 24 Well Cell Migration Assay 24 samples 100 3900-500-05 10X REC Buffer 5 1 ml 51 3465-096-K Cultrex® 96 Well Cell Migration Assay 96 samples 100 3900-500-06 10X REC Buffer 6 1 ml 48, 58, 61 3470-096-02 Sulforaphane 15 µl 97 3900-500-07 10X REC Buffer 7 1 ml 45 3470-096-K Cultrex® In Vitro Angiogenesis Assay Kit 3900-500-09 10X REC Buffer 9 1 ml 60 (***formerly HT CAS Tube Formation Kit***) 96 Tests 97 3900-500-10 10X REC Buffer 10 1 ml 50 3471-096-K Cultrex® In Vitro Angiogenesis 3900-500-11 10X REC Buffer 11 1 ml 52, 54 Assay Endothelial Cell Invasion Kit 96 tests 98 3900-500-12 10X REC Buffer 12 1 ml 47 3475-024-K Cultrex® 24 Well In Vitro Vascular 3900-500-15 10X REC Buffer 15 1 ml 64 Permeability Kit 24 tests 99 3950-010-03 REC Dilution Buffer 10 ml 31 3475-096-K Cultrex® 96 Well In Vitro Vascular 3950-040-01 25X FLARE Buffer 1 40 ml 31 Permeability Kit 96 Tests 99 3950-075-02 FLARE Slides 25 Slides 30, 31 3480-024-01 24 Well Cell Invasion Chamber 2 plates 100 3950-075-SP FLARE Sample Prep 75 Tests 31 3480-024-K CultreCoat® 24 Well Cell Invasion Assay 24 tests 100 3950-100-04 FLARE Buffer BSA Additive 100 µl 31 3481-024-K CultreCoat® 24 Well Cell Invasion 3950-100-05 FLARE Buffer Cation Solution 100 µl 31 Assay Low Range 25 tests 100 3950-300-02 FLARE Slides 100 Slides 31 3481-096-K CultreCoat® 96 Well Cell Invasion Assay Low Range 96 tests 100 3951-040-01 25X FLARE Buffer 2 40 ml 35 ® 4000-500-EB E. coli MutY Enzyme & Buffer 500 Units 62

PRICE LIST 3482-024-K CultreCoat 24 Well Cell Invasion Assay Medium Range 26 tests 100 4018-250 REC 5X Loading Buffer 250 µl 46 3482-096-K CultreCoat® 96 Well Cell Invasion 4019-1 REC water 1 ml 46 188 Assay Medium Range 96 tests 100 4020-01K-EB Human ß Polymerase Enzyme & Buffer 1000 Units 46 3483-024-K CultreCoat® 24 Well Cell Invasion 4020-100-EB Human ß Polymerase Enzyme & Buffer 100 Units 46 Assay High Range 27 tests 100 4020-100-K Human ß Polymerase Kit 100 Units 46 3483-096-K CultreCoat® 96 Well Cell Invasion 4020-500-EB Human ß Polymerase Enzyme & Buffer 500 Units 46 Assay High Range 96 tests 100 4025-100-EB E. coli Uracil N Glycosylase 3484-024-K CultreCoat® 24 Well Cell Invasion Enzyme & Buffer 100 Units 58 Optimization Assay Sampler 28 tests 100 4040-100-EB E. coli Fpg Enzyme & Buffer 500 Units 50 3484-096-K CultreCoat® 96 Well Cell Invasion Optimization Assay Sampler 96 tests 100 4040-100-FK E. coli Fpg. FLARE Kit 75 Samples 32 3490-096-01 CultreCoat® 2X CA Assay Buffer 100 ml 102 4040-100-FM E. coli Fpg FLARE Module >100 Samples 32 3490-096-02 CultreCoat® 10X Blocking Agent 3 ml 102 4040-500-EB E. coli Fpg Enzyme & Buffer 2500 Units 50 3490-096-K CultreCoat® BME 96 Well 4045-01K-EB E. coli Endonuclease III Enzyme & Buffer 1000 Units 49 Cell Adhesion Assay 96 samples 102 4045-01K-FK E. coli Endonuclease III FLARE Kit >75 Samples 34 3490-096-P CultreCoat® BME 96 Well CA Plate 1 plate 102 4045-01K-FM E. coli Endonuclease III FLARE Module >100 Samples 34 3491-096-K CultreCoat® Laminin I 96 Well 4045-05K-EB E. coli Endonuclease III Enzyme & Buffer 5000 Units 49 Cell Adhesion Assay 96 samples 102 4050-100-EB E. coli Endonuclease IV Enzyme & Buffer 100 Units 56 3491-096-P CultreCoat® Laminin I 96 Well 4050-500-EB E. coli Endonuclease IV Enzyme & Buffer 500 Units 56 CA Plate 1 plate 102 4055-100-EB T4 Endonuclease V Enzyme & Buffer 100,000 Units 52 3492-096-K CultreCoat® Collagen I 96 Well 4055-100-FK T4-PDG FLARE Kit 75 Samples 36 Cell Adhesion Assay 96 samples 102 4055-100-FM T4-PDG FLARE Module >100 Samples 36 3492-096-P CultreCoat® Collagen I 96 Well CA Plate 1 plate 102 4065-100-EB Chlorella Virus Pyrimidine Dimer Glycosylase E&B 1,000 Units 54 3493-096-K CultreCoat® Collagen IV 96 Well Cell Adhesion Assay 96 samples 102 4065-100-FK cv-PDG FLARE Kit 75 Samples 37 3493-096-P CultreCoat® Collagen IV 96 Well 4065-100-FM cv-PDG FLARE Module >100 Samples 37 CA Plate 1 plate 102 4070-500-EB Thermostable TDG Enzyme & Buffer 500 Units 63 3494-096-K CultreCoat® Fibronectin 96 Well 4090-100-EB Mouse 3-mA DNA Glycosylase Cell Adhesion Assay 96 samples 102 (Aag Protein) 100 Units 60 3494-096-P CultreCoat® Fibronectin 96 Well 4090-500-EB Mouse 3-mA DNA Glycosylase CA Plate 1 plate 102 (Aag Protein) 500 Units 60 3495-096-K CultreCoat® Vitronectin 96 Well 4100-100-EB S. pombe UVDE Enzyme & Buffer 100 µl 51 Cell Adhesion Assay 96 samples 102 4100-100-FK S. pombe UVDE FLARE Kit 75 Samples 35 3495-096-P CultreCoat® Vitronectin 96 Well 4100-100-FM S. pombe UVDE FLARE Module >100 Samples 35 CA Plate 1 plate 102 4110-01K-EB Human AP Endonuclease & Buffer 1000 Units 45 3496-096-K CultreCoat® Adhesion Protein Array Kit 96 samples 102 3496-096-P CultreCoat® Adhesion Protein Array Plate 1 plate 102

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CATALOG # DESCRIPTION SIZE LIST PRICE PAGE CATALOG # DESCRIPTION SIZE LIST PRICE PAGE 4110-05K-EB Human AP Endonuclease & Buffer 5000 Units 45 4253-096-03 96-Well CometSlide 1 Slide 30 4120-100-EB Human Fen-1 Enzyme and Buffer 100 Units 47 4253-096-ESK CometAssay® 96 Well ES Starter Kit 1 System 28 4125-100-EB Mismatched Uracil Gylcosylase 4253-096-ESK-PS1 Enzyme/Buffer 100 Units 61 CometAssay® 96 Well ES Starter Kit 4130-100-EB hoGG1 Enzyme & Buffer 100 Units 48 plus Power Supply for North America - 28 4130-100-FK hoGG1 FLARE Kit 75 Samples 33 4253-096-ESK-PS2 CometAssay® 96 Well ES Starter Kit 4130-100-FM hoGG1 FLARE Module 100 Samples 33 plus Power Supply for EU 4130-500-EB hoGG1 Enzyme & Buffer 500 Samples 48 (except UK & Switzerland) - 28 4135-250-01 Human Ku70/Ku80 250 Units 65 4253-096-ESK-PS3 4150-010-02 100 mM MgCl2 1 ml 64 CometAssay® 96 Well ES Starter Kit 4150-010-03 50 mM MnCl2 1 ml 64 plus Power Supply for UK - 28 4150-010-EB DPO-4+ Buffer + Cations 10 µg 64 4253-096-ESK-PS4 CometAssay® 96 Well ES Starter Kit 4150-050-EB DPO-4+ Buffer + Cations 50 µg 64 plus Power Supply for Switzerland - 28 4250-010-01 CometAssay® Lysis Solution 100 ml 24 4253-096-K CometAssay® Kit 96 wells 96 samples 24 4250-004-03 CometSlide 2 Slides 30 4254-200-01 20X Staining Reagent #1 1.2 ml 26 4250-050-01 CometAssay® Lysis Solution 2 x 500 ml 24 4254-200-02 20X Staining Reagent #2 1.2 ml 26 ®

4250-050-02 CometAssay LMAgarose 15 ml 24 PRICE LIST 4254-200-03 20X Staining Reagent #3 1.2 ml 26 4250-050-03 CometSlide 25 Slides 30 4254-200-04 2X Staining Reagent #4 1.2 g 26 4250-050-04 200 mM EDTA, pH 10 12.5 ml 24 4254-200-05 10X Fixation Additive 2.2 ml 26 4250-050-05 SYBR Green 5 µl 24 4254-200-K CometAssay® Silver Staining Kit 200 Samples 26 4250-050-ES CometAssay® Electrophoresis System 1 System 28 4256-010-CC CometAssay® Control Cells 10 assays 29 4250-050-ESK CometAssay® Electrophoresis Starter Kit 4257-010-NC Neutral Comet Assay Control Cells 10 assays 29 (includes CometAssay Kit, Control Cells & ES) - 28 4335-AMC-050 PAR Monoclonal Antibody Affinity Purified 50 µg/50 µl 19 4250-050-ESK-PS1 CometAssay® Electrophoresis Starter Kit 4335-MC-01K-AC 189 plus Power Supply for North America - 28 Anti-PAR Monoclonal Antibody 1000 µl 19 4250-050-ESK-PS2 4335-MC-100 Anti-PAR Monoclonal Antibody CometAssay® Electrophoresis Starter Kit w/o Control 100 µg/100 µl 19 plus Power Supply for EU 4335-MC-100-AC (except UK & Switzerland) - 28 Anti-PAR Monoclonal Antibody 4250-050-ESK-PS3 w/Control 100 µl 19 CometAssay® Electrophoresis Starter Kit 4336-100-01 Poly(ADP) Ribose (PAR) Polymer 100 µl 17 plus Power Supply for UK - 28 4336-APC-50 PAR Polyclonal Antibody 4250-050-ESK-PS4 Affinity Purified 50 µl 20 CometAssay® Electrophoresis Starter Kit 4336-BPC-100 Anti-PAR Polyclonal Antibody (Rabbit) 100 µl 20 plus Power Supply for Switzerland - 28 4338-MC-50 Anti-PARP Monoclonal Antibody 4250-050-K CometAssay® Kit 50 Samples 24 (Clone C2-10) 50 µg/50 µl 18 4250-200-03 CometSlide 100 Slides 30 4350-MC-100 Anti-UVssDNA Monoclonal Antibody 100 µg/100 µl 43 4250-500-02 CometAssay® LMAgarose 100 ml 24 4354-MC-050 Anti-8-oxo-dG Monoclonal Antibody 4251-050-K CometAssay® Silver Kit 50 Samples 26 (Clone 2E2) 50 µl 40,172 4252-02K-01 CometAssay® HT Slide 100 Slides 30 4360-MC-100 Anti-BPDE Monoclonal Antibody 4252-040-01 CometAssay® HT Slide 2 Slides 30 (Clone 8E11) 100 µg/100 µl 42 4252-040-02 CometSlide® Rack System Each 30 4370-096-K 8-oxo-dG Elisa Kit 96 samples 171 4252-040-ESK CometAssay® 20 Well ES Starter Kit 1 System 28 4410-PC-100 Anti-Fen-1 Polyclonal Antibody 100 µl 41 4252-040-ESK-PS1 4411-PC-020 Anti-Phosphorylated Histone CometAssay® 20 Well ES Starter Kit H2AX Sample 20 µl 39 plus Power Supply for North America - 28 4411-PC-100 Anti-Phosphorylated Histone - 4252-040-ESK-PS2 H2AX Polyclonal Antibody 100 µl 39 CometAssay® 20 Well ES Starter Kit 4500-10-P PARP Treated Protein Control plus Power Supply for EU for Western Blot 10 µl 19 (except UK & Switzerland) - 28 4520-096-K PARP in vivo Pharmacodynamic 4252-040-ESK-PS3 Assay 2nd Generation 96 Samples 12 CometAssay® 20 Well ES Starter Kit 4667-250-EB Recombinant Human PARP Enzyme 250 µl 15 plus Power Supply for UK - 28 4667-250-01 Recombinant Human PARP Enzyme 250 µl 15 4252-040-ESK-PS4 4667-50-01 Recombinant Human PARP Enzyme 50 µl 15 CometAssay® 20 Well ES Starter Kit plus Power Supply for Switzerland - 28 4667-50-02 10X PARP Buffer 700 µl 15 4252-040-K CometAssay® HT Sample Kit 40 Samples 24 4667-50-03 200 mM 3-Aminobenzamide 60 µl 23 4252-200-01 CometAssay® HT Slide 10 Slides 30 4667-50-06 Activated DNA for PARP Assay 500 µl 9 4252-500-01 CometAssay® HT Slide 25 Slides 30 4667-50-09 4-Amino-1,8-naphthalimide (800 µM) 100 µl 23 4667-50-10 6(5H)-Phenanthridinone (160 µM) 100 µl 23

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CATALOG # DESCRIPTION SIZE LIST PRICE PAGE CATALOG # DESCRIPTION SIZE LIST PRICE PAGE 4667-50-11 Benzamide (8 mM) 100 µl 23 4810-30-02 TACS 2 TdT Labeling Buffer 100 ml 123 4667-50-EB Recombinant Human PARP Enzyme 50 µl 15 4810-30-03 TACS 2 TdT Stop Buffer 100 ml 123 4668-100-01 Recombinant Human PARP 4810-30-04 TACS 2 TdT dNTP 30 µl 123 Enzyme (HSA) 1,000 Units 15 4810-30-05 TdT Enzyme 30 µl 123 4668-02K-01 Recombinant Human PARP 4810-30-10 Magnesium Cation 30 µl 123 Enzyme (HSA) 20,000 Units 15 4810-30-CI Cations (1 of each) 30 µl each 123 4668-500-01 Recombinant Human PARP 4810-30-CK TACS 2 TdT-Core Kit 30 Samples 123 Enzyme (HSA) 5,000 Units 15 4810-30-K TACS 2 TdT-DAB Kit 30 Samples 123 4670-500-01 Biotinylated NAD+ 500 µl 21 4810-30-R TdT 2 Replenisher Kit 30 Samples 123 4671-096-02 20X PARP Buffer 500 µl 9 4810-90-09 Cobalt Cation 3 x 30 µl 123 4671-096-03 10X PARP Cocktail 300 µl 9 4810-90-10 Magnesium Cation 3 x 30 µl 123 4671-096-04 10X Strep-Diluent 2.0 ml 9 4810-90-14 Manganese Cation 3 x 30 µl 123 4671-096-06 10X Activated DNA 300 µl 15 4811-30-K TACS 2 TdT-Blue Label Kit 30 Samples 123 4673-500-01 Fluorescein-NAD+ 250 µl 22 4812-30-K TACS 2 TdT-Fluorescein Kit 30 Samples 123 4674-250-01 Cell Permeabilization Solution 250 µl 22 4815-30-K TumorTACS Kit 30 Samples 132 4675-096-01 PeroxyGlow A 6 ml 9,10,12,14 4817-60-02 10X TdT Labeling Buffer 20 ml 135,137 4675-096-02 PeroxyGlow B 6 ml 9,10,12,14 4817-60-03 10X TdT Stop Buffer 20 ml 135,137 4676-096-K Universal Chemiluminescent PARP Assay Kit w/ Histone Coated Strip wells 96 Samples 9 4817-60-04 PI/RNase 1 ml 137 4677-096-K Universal Colorimetric PARP 4817-60-K FlowTACS Kit 60 Samples 137 Assay Kit w/ Histone Coated Strip Wells 96 Samples 9 4820-30-01 NeuroPore 5 ml 157 4677-096-P 96-Well Histone Coated Plate 4820-30-13 Blue Counterstain 50 ml 156 (stripwell) (natural) 1 plate 9, 14 4820-60-01 NeuroPore 2 x 5ml 157 PRICE LIST 4678-096-P 96-Well Histone Coated Plate 4821-96-01 Proteinase K 100 µl 135 (stripwell) (white) each 9, 14 4821-96-04 TdT dNTP 35 µl 135 4680-096-01 PARG Enzyme (1µg/mL) 300 µl 16 190 4821-96-05 TdT Enzyme 35 µl 135 4680-096-02 10X PARG Buffer 8 mL 14, 16 4821-96-14 Manganese Cation 100 µl 135 4680-096-03 100 mM DEA 200 µl 14, 23 4822-96-08 TACS-Sapphire 10 ml 9,10,14,135 4682-096-K HT Chemiluminescent PARG Assay Kit 96 Tests 14 4822-96-K TiterTACS Colorimetric Kit 96 Samples 135 4683-096-K HT Colorimetric PARG Assay Kit 96 Tests 14 4823-30-K NeuroTACS II Kit 30 Samples 130 4684-096-02 20mM NAD+ 300 µl 10 4826-30-K VasoTACS Kit 30 Samples 134 4684-096-03 5X Antibody Diluent 3 ml 10 4827-30-K CardioTACS Kit 30 Samples 126 4684-096-05 HRP Conjugate 20 µl 10 4828-30-04 TACS B-dNTP Mix 30 µl 121 4684-096-K HT Colorimetric PARP Apoptosis 4828-30-06 anti-BrdU antibody 30 µl 121 Assay Kit 96 Tests 10 4828-30-12 Strep-Diluent 7.5 ml 121 4685-096-K HT Chemiluminescent PARP Apoptosis Assay Kit 96 Tests 10 4828-30-18 Methyl Green 50 ml 121 4690-096-K HT F Homogeneous PARP 4828-30-AC TACS.XL Antibody Module 30 Samples 121 Inhibition Assay Kit 96 Tests 11 4828-30-BC TACS Blue Label Detection Module 30 Samples 121 4800-30-01 Proteinase K 30 µl 157 4828-30-BK TACS.XL Blue Labeling Kit 30 Samples 121 4800-30-06 Streptavidin-HRP 30 µl 158 4828-30-DC TACS.XL DAB Detection Module 30 Samples 121 4800-30-07 Diaminobenzidine 3.75 ml 121-124 4828-30-DK TACS.XL DAB Kit 30 Samples 121 4800-30-08 Methyl Green Stain, 0.5% 50 ml 156 4828-30-K TACS.XL Kit 30 Samples 121 4800-30-11 TACS Blue Label 3 ml 122-124 4828-30-N TACS.XL Nuclease Module 15 Samples 121 4800-30-12 Blue Strep-HRP Diluent 7.5 ml 122-124 4828-30-R TACS.XL Replenisher Kit 30 Samples 121 4800-30-14 Streptavidin-FITC 30 µl 158 4829-30-K DermaTACS Kit 30 Samples 128 4800-30-17 Nuclear Fast Red 50 ml 156 4830-01-1 Annexin V FITC 100 µl 118 4800-30-18 Methyl Green 1% 50 ml 156 4830-01-2 10X Binding Buffer 8 ml 118 4800-30-19 Red Counterstain C 50 ml 156 4830-01-3 Propidium Iodide 1 ml 118 4800-30-20 Cell Control Slides 2 each 156 4830-01-K Annexin V FITC Kit 100 Samples 118 4800-30-21 Cell Control Slides 5 each 156 4830-250-1 Annexin V FITC 250 µl 118 4800-30-40 Tissue Control Slides 2 each 156 4830-250-2 10X Binding Buffer 20 ml 118 4800-30-41 Tissue Control Slides 5 each 156 4830-250-3 Propidium Iodide 2.5 ml 118 4800-30-42 EpiDerm Control Slides 2 each 156 4830-250-K Annexin V FITC Kit 250 Samples 118 4800-30-BC Blue Label Conversion Kit 30 Samples 121 4835-01-1 Annexin V Biotin 100 µl 120 4800-30-BL Blue Label and Diluent 4835-01-K Annexin V Biotin Kit 100 Samples 120 (4800-30-11 & 4800-30-12) 30 Samples 122 4835-250-1 Annexin V Biotin 250 µl 120 4800-30-N TACS Nuclease and Buffer 15 µl 156 4835-250-K Annexin V Biotin Kit 250 Samples 120 4850-20-01 Lysis Solution 1 2 x 1 ml 181

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Trevigen Price List

CATALOG # DESCRIPTION SIZE LIST PRICE PAGE CATALOG # DESCRIPTION SIZE LIST PRICE PAGE 4850-20-02 Extraction Solution 2 20 ml 181 5511-001-01 MPG Knock Down Cell Line 1 vial 5 4850-20-03 Extraction Buffer 3 8 ml 181 5512-001-01 MultYH Knock Down Cell Line 1 vial 5 4850-20-04 Sodium Acetate 4 1 ml 181 5513-001-01 Neil1 Knock Down Cell Line 1 vial 5 4850-20-05 DNase-Free Water 5 2 ml 181 5514-001-01 PARP2 Knock Down Cell Line 1 vial 5 4850-20-06 Sample Buffer 2 ml 181 5515-001-01 PARP3 Knock Down Cell Line 1 vial 5 4850-20-10 Gel Loading Buffer 250 µl 139 5516-001-01 ERCC1 Knock Down Cell Line 1 vial 5 4850-20-ET Apoptotic DNA Laddering Kit, EtBr 20 Samples 139 5517-001-01 APE1 Knock Down Cell Line 1 vial 5 4850-20-GD Genomic DNA Isolation Kit 20 Samples 181 5518-001-01 APE2 Knock Down Cell Line 1 vial 5 4859-20-01 10X Tissue Buffer 500 µl 181 5519-001-01 TDG Knock Down Cell Line 1 vial 5 4859-20-K Apoptotic DNA Laddering Kit 6300-100-01 DePsipher 100 µl 140 Tissue Supplement 20 Samples 181 6300-100-02 10X Reaction Buffer 2 x 30 ml 140 4861-100 Treated Glass Microscope Slides 100 Slides 157 6300-100-03 Stabilizer Solution 5 ml 140 4862-10 Glass Coverslips, 22 x 60 mm 10 oz 157 6300-100-K DePsipher Kit 100 Tests 140 4864-100 3 Ring Treated Glass Microscope Slides 100 Slides 157 6305-100-01 MitoShift (1mM) 100 µl 142 4865-25 Mounting Medium 25 ml 156 6305-100-02 Valinomycin 100 µl 156 4866-20 Fluorescent Mounting Medium 20 ml 156 6305-100-K MitoShift Kit 100 Tests 142

4867-100 Hydrophobic Coverslips 100 Each 157 6360-025-01 PBR Protein (Recombinant) 25 µl 153 PRICE LIST 4868-500-06 25X PBS 6 x 500 ml 183 6361-PC-100 Anti-Human/Mouse PBR Antibody 100 µl 153 4869-500 Apoptosis Grade Water 500 ml 156 6362-PC-100 Anti-Mouse PBR Antibody 100 µl 153 4869-500-6 Apoptosis Grade Water 6 x 500 ml 156 7500-100-01 SOD (1unit/µl) 200 µl 174 4870-500 10X PBS 500 ml 156 7500-100-02 25X SOD Reaction Buffer 12 ml 174 4870-500-6 10X PBS Buffer, pH 7.4 6 x 500 ml 156 7500-100-03 Xanthine Solution 1.5 ml 174 4876-05-01 Cytonin 5 ml 157 7500-100-04 NBT Solution 6.0 ml 174 4876-05-02 Cytonin 2 x 5 ml 157 7500-100-05 XOD Solution 2.0 ml 174 4878-05-01 Cytonin IHC 5 ml 157 7500-100-06 20X Cell Lysis Solution 12.0 ml 174 191 4878-05-02 Cytonin IHC 2 x 5 ml 157 7500-100-K SOD Assay Kit 100 reactions 174 4886-100-02 Staurosporine 100 µl 156 7501-500-03 Xanthine Oxidase 3 ml 173 4886-400-01 Etoposide 400 µl 156 7501-500-K HT Superoxide Dismutase Assay Kit 480 tests 173 4887-100-03 Streptavidin-AMCA 100 µl 158 7510-100-01 Glutathione Reductase 500 µl 178 4890-25-01 MTT Reagent 25 ml 145 7510-100-02 25X Glutathione Reductase 4890-25-02 Detergent Reagent 250 ml 145 Reaction Buffer 20 ml 178 4890-25-K MTT Cell Proliferation Assay Kit 2500 Tests 145 7510-100-03 NADPH 10 x 9.2mg 178 4890-50-K MTT Cell Proliferation Assay 5000 Tests 145 7510-100-04 GSSG Solution 10 ml 178 4891-025-01 XTT Reagent 5 x 25 ml 146 7510-100-05 25X Sample Dilution Buffer 20 ml 178 4891-025-02 XTT Activator 5 x 500 µl 146 7510-100-06 25X Tissue Homogenization Buffer 40 ml 178 4891-025-K XTT Cell Proliferation Assay Kit 2500 Tests 146 7510-100-K Glutathione Reductase Assay 100 Reactions 178 4892-010-01 Calcein AM 50 µg 147 7511-100-01 Glutathione Reductase 80 µl 175 4892-010-02 10X Calcein AM DW Buffer 200 ml 147 7511-100-02 25X Assay Buffer 12 ml 175 4892-010-K Calcein AM Cell Viability Assay Kit 1000 Tests 147 7511-100-04 Reaction Mix 8 Bottles 175 5000-001-01 Rat Mesenchymal Cells 1 vial 166 7511-100-05 96-Well Plates 8 Plates 175 5000-001-K Rat Mesenchymal Stem Cell Starter Kit 24 wells 166 7511-100-K HT Glutathione Assay Kit 384 Tests 175 5000-001-R Rat Mesenchymal Replenisher Kit 500/50 ml 166 7512-100-01 Glutathione Peroxidase 800 µl 176 5010-024-K Mesenchymal Stem Cell Adipogenic 7512-100-02 10X Assay Buffer 20 ml 176 Differentiation Kit 24 wells 167 7512-100-03 GSH+NADPH 10 Vials 176 5011-024-K Mesenchymal Stem Cell Osteogenic 7512-100-04 Glutathione Reductase 1.1 ml 176 Differentiation Kit 24 wells 168 7512-100-05 Cumene Hydroperoxide 12 ml 176 5500-001-01 PARP1 Knock Down Cell Line 1 vial 5 7512-100-06 96-well plates 5 Plates 177 5501-001-01 PARG Knock Down Cell Line 1 vial 5 7512-100-K HT Glutathione Peroxidase Assay Kit 480 Tests 176 5502-001-01 BRCA1 Knock Down Cell Line 1 vial 5 7513-500-K HT Glutathione Reductase Assay Kit 480 Tests 177 5503-001-01 Knock Down Control Cell Line 1 vial 5 9510-01K-EB Terminal Deoxynucleotidyl Transferase 1000 Units 184 5504-001-01 OGG1 Knock Down Cell Line 1 vial 5 9600-5-D Calf Thymus DNA 5 x 1 ml 184 5505-001-01 NTHL1 Knock Down Cell Line 1 vial 5 9605-5-D Herring Sperm DNA 5 x 1 ml 184 5506-001-01 MBD4 Knock Down Cell Line 1 vial 5 9610-5-D Salmon Sperm DNA 5 x 1 ml 184 5507-001-01 NEIL2 Knock Down Cell Line 1 vial 5 9804-050-P TreviGel 500 50 g 182 5508-001-01 NEIL3 Knock Down Cell Line 1 vial 5 9804-100-P TreviGel 500 100 g 182 5509-001-01 UNG Knock Down Cell Line 1 vial 5 9804-250-P TreviGel 500 250 g 182 5510-001-01 SMUG1 Knock Down Cell Line 1 vial 5 9806-050-P TreviGel 5000 50 g 182

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PRICE LIST

Trevigen Price List

CATALOG # DESCRIPTION SIZE LIST PRICE PAGE CATALOG # DESCRIPTION SIZE LIST PRICE 9806-100-P TreviGel 5000 100 g 182 C1000 Catalog Each 9806-250-P TreviGel 5000 250 g 182 MPCM-1000 ApoMug Each 9850-250-6 5X Orange G Loading Buffer 6 x 250 µl 183 MPCM-1001 DNA Damage Mug Each 9860-500 50X TAE Electrophoresis Buffer 500 ml 183 MPP3-1000 3D Assay Poster Each 9860-500-2 50X TAE Electrophoresis Buffer 2 x 500 ml 183 MPPI-1000 Cell Invasion Poster Each MPPT-1000 Tube Assay Poster Each

Appendix PRICE LIST / APPENDIX DNA DAMAGE APOPTOSIS DETECTION 192 PARP Inhibitor screening ...... 9, 10, 11, 12, 15 Light Microscopy ...... 25, 121-134, 145, 146 PARP Activity detection ...... 9, 10, 15 Fluorescent Microscopy . . .25, 118, 120, 123, 140, 142, 145, 146 PARG Inhibition screening ...... 16 Flow Cytometry ...... 118, 120, 137, 140-142 PARG Activity detection ...... 14, 16 Gel Electrophoresis ...... 25, 139 Double/Single DNA Strand Break analysis ...... 24, 26 Plate Reader ...... 135, 144-147 DNA Repair Inhibitors screening ...... 24, 26 Double Label ...... 121-137 DNA Repair Gene Knockdown Cell lines ...... 5 Confocal Microscopy ...... 118, 120, 123, 140-142 Cellular Response to DNA Damage ...... 24, 26 Unfixed Cells ...... 25, 118, 120, 139-147 Toxicity testing ...... 24, 26 Fixed Cells ...... 121-137 Specific DNA Damage Type probing ...... 31-37 Paraffin Embedded Tissue ...... 121-139 Fresh Tissue ...... 120, 139, 144 OXIDATIVE DAMAGE In Situ Apoptosis Detection ...... 118-138 Enzymatic Assays ...... 173-178 Cell Proliferation ...... 145, 146 ELISA (8-oxo-dG) ...... 171, 172 Cell Viability ...... 147 Immunocytochemistry ...... 172 Cytotoxicity analysis ...... 145-147

CANCER CELL BEHAVIOR STEM CELL CULTURE, DIFFERENTIATION 3-D Culture ...... 76, 105-110 Stem Cells ...... 166 Cell Invasion and Migration ...... 98-100 Differentiation Kits ...... 167, 168 Adhesion and Attachment ...... 72, 80-91, 102 Stem Cell-qualified Reagents ...... 165 Tube formation ...... 72, 97 Angiogenesis Assay ...... 72, 95, 97, 98 Clonogenic Assays ...... 70, 72 Subcutaneous “Plug” Assay ...... 72, 74 Tumorgraft and Xenograft Assays ...... 72, 74

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INDEX

Product Index

C D

3-D Culture BME Cell Calcein AM Cell Viability Assay Kit ...... 147 DAB Solution ...... 121 Proliferation Assay Kit ...... 108 Calf Thymus DNA ...... 184 DEA ...... 23 3-D Culture Cell Harvesting Kit ...... 109 Cancer Cell Assays ...... 94 DePsipher™ Kit ...... 140 3-D Culture Cell Proliferation Reagent ...... CardioTACS™ Kit ...... 126 DermaTACS™ Kit ...... 128 3-D Culture Collagen I ...... 107 Cell Assays Services ...... 186 Detergent Reagent (for MTT assay) . . . . .145 3-D Culture Collagen I Cell Cell Culture Control Slides ...... 156 DIVAA™ Kits and Components ...... 95 Proliferation Assay Kit ...... 108 Cell Invasion BME Assay Kit, Cultrex® . . .100 DNA (Apoptotic) Laddering Kit ...... 139 3-D Culture Laminin I ...... 106 Cell Invasion Collagen I Assay Kit, DNA (Genomic) Isolation Kit ...... 181 3-D Culture Laminin I Cell Cultrex® ...... 100 DNA Polymerase β (human) ...... 46 Proliferation Assay Kit ...... 108 Cell Invasion Collagen IV Assay Kit, DNA Polymerase β (human) kit ...... 46 3-D Culture Matrix™ (BME) ...... 105, 76 Cultrex® ...... 100 DNA Repair Gene Knockdown Cell Lines . . .5 ®

8-oxo-dG Elisa Kit ...... 171 Cell Migration Assay, Cultrex ...... 100 Dpo4 Enzyme ...... 64 PRODUCT INDEX 8-oxo-dG Monoclonal Antibody . . . . .40, 172 Cell Permeabilization Solution ...... 22 Cell Proliferation Assay Kit, MTT/XTT . .146,145 E A Cell Staining Kit, Cultrex® ...... 104 Cleaved-Caspase-3 Polyclonal Antibody . .154 Electrophoresis buffer ...... 183 Aag (mouse) Enzyme ...... 60 Collagen I (bovine), Cultrex® ...... 87 Endonuclease III (E. coli) Enzyme ...... 49 Activated DNA ...... 15 Collagen I (rat), Cultrex® ...... 85 Endonuclease IV (E. coli) Enzyme ...... 56 Angiogenesis Assay Direct Collagen IV (mouse), Cultrex® ...... 86 Enzymes, DNA Repair ...... 44 (In Vivo) (DIVAA™) Kit ...... 95 CometAssay® Control Cells EpiDerm™ Control Slides ...... 128 Angiogenesis Assay Endothelial Cell (alkaline/neutral) ...... 29 Etoposide ...... 156 Invasion Kit (In Vitro), Cultrex® ...... 98 CometAssay®Electrophoresis Starter Kit . .28 193 Angiogenesis Assay Tube Formation CometAssay® Electrophoresis Unit ...... 28 F Kit (In Vitro), Cultrex® ...... 97 CometAssay® FLARE™ Kits ...... 31 AngioRack™ ...... 95 CometAssay® Kit ...... 24 FEN-1 (human) Enzyme ...... 47 Annexin V Biotin Kit ...... 120 CometAssay® LMAgarose ...... 24 FEN-1 Polyclonal Antibody ...... 41 Annexin V FITC Kit ...... 118 CometAssay® Lysis Solution ...... 24 Fibronectin, Cultrex® ...... 88 Antibodies, Apoptosis ...... 148 CometAssay® Silver Kit ...... 26 FLARE™ Kit, cv-PDG ...... 37 Antibodies, DNA Damage ...... 38 CometAssay® Silver Staining Kit ...... 26 FLARE™ Kit, Endonuclease III ...... 34 Apoptosis Hints, Tips, Protocols ...... 156 CometAssay® Staining Reagents ...... 26 FLARE™ Kit, Fpg ...... 32 Apoptosis In Situ Kits ...... 117 CometSlide™ ...... 30 FLARE™ Kit, hOGG1 ...... 33 Apoptosis Stains, Buffers, Accessories . .156 CometSlide™ Rack System ...... 30 FLARE™ Kit, T4-PDG ...... 36 Apoptotic DNA Laddering Kit ...... 139 Counterstains (Histological) ...... 156 FLARE™ Kit, UVDE ...... 35 CultreCoat® Adhesion Protein Array Kit . .102 FLARE™ Slides ...... 30 B CultreCoat® BME Cell Adhesion Assay . . .102 FlowTACS™ Kit ...... 137 CultreCoat® BME Cell Invasion Assays . . .100 Fluorescein-NAD+ ...... 22 Bax Monoclonal Antibody ...... 149 CultreCoat® Cell Invasion Fluorescent Mounting Medium ...... 156 Bcl-2 Monoclonal Antibody ...... 151 Optimization Assays ...... 100 Fpg (E. coli) Enzyme ...... 50 Benzamide ...... 23 CultreCoat® Collagen I Cell BETA Polymerase (human) ...... 46 Adhesion Assay ...... 102 G BETA Polymerase (human) Kit ...... 46 CultreCoat® Collagen IV Cell Blue Counterstain ...... 156 Adhesion Assay ...... 102 G3PDH Polyclonal Antibody ...... 155 BME High Protein Concentration, Cultrex® .74 CultreCoat® Fibronectin Cell Glass Coverslips ...... 157 BME Human, Cultrex® ...... 80 Adhesion Assay ...... 102 Glutathione Assay Kit ...... 175 BME PathClear®, Cultrex® ...... 72 CultreCoat® Laminin I Cell Glutathione Peroxidase Assay Kit ...... 176 BME Stem Cell Qualified, Cultrex® . . .75, 165 Adhesion Assay ...... 102 Glutathione Reductase Assay Kit . . .177, 178 BME Mouse, Cultrex® ...... 70 CultreCoat® Vascular Permeability BPDE Monoclonal Antibody ...... 42 (In Vitro) kits ...... 99 H BrdU Antibody ...... 128 CultreCoat® Vitronectin Cell BrdU-dNTP mix ...... 128 Adhesion Assay ...... 102 hAPE Enzyme ...... 45 Biotin - NAD+ ...... 21 Cultrex® Products ...... 69 Herring Sperm DNA ...... 184 cv-PDG Enzyme ...... 54 Histone Coated Strip Wells (natural/white) . .9 Cytonin- IHC™ ...... 157 hoGG1Enzyme ...... 48 Cytonin™ ...... 157 Human BME PathClear® ...... 80

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INDEX

Product Index

Human DNA Polymerase Beta ...... 46 PARG Enzyme ...... 16 TCA (Trevigen Cell Assays) Services . . . . .186 Human Fen-1 ...... 47 PARP Apoptosis Assay Kit ...... 10 TDG Enzyme ...... 63 Human Fibronectin PathClear® ...... 81 (Chemiluminescent/Colorimetric) TdT Buffers ...... 117 Human Ku70/80 ...... 65 PARP Enzyme and Buffer ...... 15 TdT Enzyme ...... 184 Human PARP ...... 15 PARP Enzyme High Specific Activity ...... 15 Technical Resources ...... 195 Human Vitronectin PathClear® ...... 82 PARP Inhibition Assay Kit, Tissue Control Slides ...... 156 Hydrophobic Cover Slips ...... 158 Fluorescein Homogeneous ...... 11 Tissue Extraction Reagents ...... 181 H2AX Antibodies ...... 39 PARP Monoclonal Antibody ...... 18 Tissue Supplement Kit ...... 139 PARP Pharmacodynamic Assay TiterTACS™ Kit ...... 135 K 2nd Generation (In Vivo) ...... 12 TreviGel™ ...... 182 PARP Treated Protein Control for TumorTACS Kit™ ...... 132 Knockdown Cell Lines ...... 5 Western Blot ...... 19 Ku 70/80 (human) Enzyme ...... 65 PARP Universal Assay Kit U w/ Histone Coated Strip Wells L (Chemiluminescent/Colorimetric) ...... 9 UNGase (E. coli) Enzyme ...... 58 PARP/PARG Assays ...... 8 UVDE (S. pombe) Enzyme ...... 51 Laminin (mouse), Cultrex® ...... 83, 84 PARP/PARG Inhibitors ...... 23 UVssDNA Monoclonal Antibody ...... 43 PBR Polyclonal Antibody ...... 153 M PBR Protein ...... 153 V PBS ...... 183 Mesenchymal Rat Replenisher Kit ...... 166 PeroxyGlow™ A&B ...... 10, 14 Valinomycin ...... 156

PRODUCT INDEX Mesenchymal Rat Stem Cell Starter Kit . .166 Phenanthridinone ...... 23 Vascular Permeability Kit Mesenchymal Rat Stem Cells ...... 166 Phenol Red Solution ...... 70 (In Vitro), Cultrex® ...... 99 194 Mesenchymal Stem Cell Adipogenic Poly-D-Lysine, Cultrex® ...... 90 VasoTACS™ Kit ...... 134 Differentiation Kit ...... 167 Poly-L-Lysine, Cultrex® ...... 91 Vitronectin, Cultrex® ...... 89 Mesenchymal Stem Cell Osteogenic Propium Iodide ...... 118, 120 Differentiation Kit ...... 168 Proteinase K ...... 184 W Methyl Green ...... 156 MitoShift™ Kit ...... 142 R Water, Apoptosis Grade™ ...... 156 Mounting Medium (toluene based) ...... 156 MTT Cell Proliferation Assay Kit ...... 145 REC Buffers ...... 44 X Mug (E. coli) Enzyme ...... 61 Red Counterstain C ...... 156 MutY (E. coli) Enzyme ...... 62 XTT Cell Proliferation Assay Kit ...... 146 S N Salmon Sperm DNA ...... 184 Naphtalimide ...... 23 Slides, Treated ...... 157 NeuroPore™ ...... 157 Staurosporine ...... 156 NeuroTACS™ II Kit ...... 130 Streptavidin ...... 158 Nuclear Fast Red ...... 156 Superoxide Dismutase Assay Kit . . .173, 174 O T

Oxidative Stress Assays ...... 169 T4-PDG (Endonuclease V) Enzyme ...... 52 TACS.XL® Blue Label Kit ...... 121 P TACS.XL® DAB Kit ...... 121 TACS.XL® Kit ...... 121 PAR Monoclonal Antibody ...... 19 TACS.XL® Replenisher Kit ...... 121 PAR Monoclonal Antibody TACS® 2 TdT Blue Label Kit ...... 123 Affinity Purified ...... 19 TACS® 2 TdT DAB Kit ...... 123 PAR Polyclonal Antibody ...... 20 TACS® 2 TdT Fluorescein Kit ...... 123 PAR Polyclonal Antibody Affinity Purified . .20 TACS® 2 TdT Replenisher Kit ...... 123 PAR Polymer ...... 17 TACS® 2 TdT-Core Kit ...... 123 PARG Assay Kit TACS® Nuclease and Buffer ...... 117 (Chemiluminescent/Colorimetric) ...... 14 TACS-Sapphire™ ...... 135

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Trevigen Peer Reviewed Publications: 1. Benton, G., George, J., Klienman, H., Arnoutoava, I. Multiple uses of basement membrane-like matrix (BME/Matrigel) in vitro and in vivo with cancer cells (Accepted). International Journal of Cancer. 2010 Nov. 2. Benton, G., George, J., Klienman, H., Arnoutoava, I. Advancing Science and Technology Via 3D Culture on Basement Membrane Matrix. J Cell Physiol. 2009 Oct; 22 1(1):18-25. 3. Benton, G., George, J. : Laminin 1 induces E-Cadherin expression in three dimensional cultured breast cancer cells by inhibiting DNA methyltransferase 1 and reversing promoter methylation status FASEB J. 2009 Nov; 23(11):3884-95. 4. Arnaoutova, I. George, J., Kleinman, H., Benton, G. The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. Angiogenesis. 2009 12(3):267-74. 5. Arnaoutova, I.P., Kleinman, H.K. In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nature Protocol. 2010 5 (4):628-35. Trevigen White Papers: 6. Klienman, H., Uses of PathClear® Basement Membrane Extract (BME): tumor growth, tumorgraft, angiogenesis, tissue transplantation, repair/regeneration, engineering, etc. 7. George, J., DNA Repair Deficient Cell Lines—Tools to study Genomic Instability. 8. George, J., The H2AX Tool Box. 9. George, J., The Need for a PARP in vivo Pharmacodynamic Assay Update 2010.

Please inquire by calling us at 1-800-TREVIGEN or e-mailing us at [email protected] For more information go to www.trevigen.com

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Notes NOTES

196

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DISTRIBUTORS

Trevigen Products International Distributors

Argentina China Hong Kong Antibody.com.Ar ChinaGen, Inc. Onwon Trading, Ltd. Tel: +54 911 3057 4190 Tel: +86 755 8631 3122; Tel: +852 2757 7569 Email: [email protected] +86 755 8631 3299 Fax: +852 2757 7211 Web: www.antibody.com.ar Fax: +86 755 8631 3266 Email: [email protected] Email: [email protected] Web: www.onwon.com.hk Australia Web: www.chinagen.com.cn Bio Scientific, Pty., Ltd. Hungary Tel: +61 2 9521 2177, 13 0024 6724 Denmark Biozol Diagnostica Fax: +61 2 9542 3100 Tel: +49 089 3799 6666 Nordic BioSite ApS Email: [email protected] Fax: +49 089 3799 66699 Tel: +46 8 5444 3340; Web: www.biosci.com.au Email: [email protected] 1800 10595 Web: www.biozol.de Fax: +46 8756 9490 Austria Email: [email protected] Szabo-Scandic HandelsgmbH Web: www.nordicbiosite.dk Iceland & Co KG Nordic BioSite AB Tel: +43 1 4893 9610 Ecuador Tel: +46 8 5444 3340 Fax: +43 1 4893 9617 Fax: +46 8756 9490 Antibody CL Email: [email protected] Email: [email protected] Tel: +56 2234 5765 Web: www.szabo-scandic.com Web: www.biosite.se Email: [email protected] Belgium Web: www.antibody.cl India Gentaur BVBA Estonia Genex Life Sciences Pvt., Ltd. Tel: +32 1658 9045 Tel: +91 98 9229 1064; +91 98 7009 1064 Fax: +32 1650 9045 Nordic BioSite AB Fax: +91 22 2612 70169 Tel: +46 8 5444 3340 DISTRIBUTORS Email: [email protected] Email: [email protected] Web: www.gentaur.com Fax: +46 8756 9490 Email: [email protected] Indonesia Web: www.biosite.se 198 Sanbio B.V. BioSynTech Sdn Bhd Tel: +02 219 2137 Finland Tel: +6 03 8025 1603 Fax: +31 4 1326 6605 Fax: +6 03 8025 1637/1354 Email: [email protected] Nordic BioSite OY Email: [email protected] Web: www.sanbio.nl Tel: 800 11 1333 Web: www.bstmgroup.com Fax: +46 8 756 9490 Brazil Email: [email protected] Ireland Web: www.biosite.fi Antibody CL AMS Biotechnology (Europe), Ltd. Tel: +56 2234 5765 France Tel: +44 0 12 3582 8200 Email: [email protected] Fax: +44 0 12 3582 0482 Web: www.antibody.cl Interchim Email: [email protected] Tel: + 33 04 7003 8855 Web: www.amsbio.com Canada Fax: + 33 04 7003 8260 Email: [email protected] Cedarlane Laboratories Ltd. Israel Web: www.interchim.com Tel: +289 288 0001; 800 268 5058 Almog Diagnostic Fax: +289 288 0020; 800 638 5099 Tel: + 972 3977 3390 Gentaur SARL Email: [email protected] Fax: + 972 3977 3391 Tel: +33 01 4325 0150 Web: www.cedarlanelabs.com Email: [email protected] Fax: +33 01 4325 0160 Web: www.almog.co.il Email: [email protected] VWR International Web: www.clonagen.com Tel: 800 932 5000 Italy Email: [email protected] Web: www.vwr.com Germany TEMA ricerca S.r.l. Tel: +39 0 5 1624 0700 Biozol Diagnostica Fax: +39 0 5 1624 0706 Trevigen, Inc. Tel: +49 089 3799 6666 Email: [email protected] Tel: 800 873 8443 Fax: +49 089 3799 66699 Web: www.temaricerca.com Fax: 301 560 4973 Email: [email protected] Email: [email protected] Web: www.biozol.de Web: www.trevigen.com Japan AMS Biotechnology GmbH Funakoshi Co., Ltd. Chile Tel: +49 0 6977 9099 Tel: +81 3 5684 1620 Fax: +49 0 69 1337 6880 Fax: +81 3 5684 1775 Antibody CL Email: [email protected] Email: [email protected] Tel: +56 2234 5765 Web: www.amsbio.com Web: www.funakoshi.co.jp Email: [email protected] Web: www.antibody.cl Gentaur Cosmo Bio Co., Ltd. Tel: +32 1658 9045 Tel: +81 5632 9610/9620 Fax: +32 1650 9045 Fax: +81 5632 9619 Email: [email protected] Email: [email protected] Web: www.bioprice.com Web: www.cosmobio.co.jp

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DISTRIBUTORS s Trevigen Products International Distributors

Korea Peru Thailand Kormed Corp. Antibody CL Truong Bio, Inc. Tel: +82 31 777 3101 Tel: +56 2234 5765 Tel: +84 8 3755 3648 Fax: +82 31 777 3999 Email: [email protected] Fax: +84 8 3755 4403 m.hk Email: [email protected] Web: www.antibody.cl Email: [email protected] hk Web: www.kormed.com Poland Turkey Latvia Gentaur Sp. Z.o.o. SitoGen BioMedikal ve Laboratuar Nordic BioSite AB Tel: +48 58 710 3344 Tel: +90 021 6489 3344 Tel: +46 8 5444 3340 Fax: +48 58 710 3348 Fax: +90 021 6489 0220 99 Fax: +46 8756 9490 Email: [email protected] Email: [email protected] Email: [email protected] Web: www.gentaur.com Web: www.sitogen.com.tr Web: www.biosite.se Biozol Diagnostica United Kingdom Tel: +49 089 3799 6666 Lithuania AMS Biotechnology (Europe), Ltd. Fax: +49 089 3799 66699 Nordic Biosite AB Tel: +44 0 12 3582 8200 Email: [email protected] Tel: +46 8 5444 3340 Fax: +44 0 12 3582 0482 Web: www.biozol.de Fax: +46 8756 9490 Email: [email protected] DISTRIBUTORS Email: [email protected] Web: www.amsbio.com Web: www.biosite.se Portugal AbBcn S.L. (AntibodyBcn) United States Tel: +34 9 3586 8985 Luxembourg Trevigen, Inc. Fax: +34 9 3581 4426 Pvt., Ltd. Sanbio B.V. Tel: 800 873 8443 Email: [email protected] +91 98 7009 1064 Tel: +3 14 1325 1115 Fax: 301 560 4973 Web: www.antibodybcn.com 9 Fax: +3 14 1326 6605 Email: [email protected] om Email: [email protected] Web: www.trevigen.com Web: www.sanbio.nl Singapore BST Scientific Pte., Ltd. VWR International Malaysia Tel: + 65 6777 2856 Tel: 800 932 5000 199 Fax: + 65 6777 2372 Web: www.vwr.com BioSynTech Sdn Bhd Email: [email protected] 354 Tel: +6 03 8025 1603 Web: www.bstsci.com com Fax: +6 03 8025 1637/1354 Fisher Scientific com Email: [email protected] Tel: 800-766-7000 Web: www.bstmgroup.com Spain Web: www.fishersci.com AbBcn S.L. (AntibodyBcn) Venezuela Europe), Ltd. Mexico Tel: +34 9 3586 8985 Fax: +34 9 3581 4426 0 Control Tecnico y Representaciones, Antibody CL Email: [email protected] Tel: +56 2234 5765 2 S.A. de C.V. (CTR Scientific) Web: www.antibodybcn.com Email: [email protected] m Tel: +52 81 8158 0600 Web: www.antibody.cl Fax: +52 81 8373 2891 Email: [email protected] Sweden Web: www.ctr.com.mx Nordic BioSite AB Vietnam Tel: +46 8 5444 3340 Truong Bio, Inc. Netherlands Fax: +46 8 756 9490 Tel: +84 8 3755 3648 Email: [email protected] Fax: +84 8 3755 4403 Sanbio B.V. Web: www.biosite.se Email: [email protected] Tel: +3 14 1325 1115 Fax: +3 14 1326 6605 Email: [email protected] Switzerland Web: www.sanbio.nl AMS Biotechnology (Europe), Ltd. Tel: +41 09 1604 5522 Fax: +41 09 1605 1785 New Zealand Email:[email protected] maricerca.com Bio Scientific, Pty., Ltd. Web: www.amsbio.com com Tel: 0800 44 4157 Fax: +61 2 9542 3100 Taiwan Email: [email protected] Bio Pioneer Tech Co., Ltd. Web: www.biosci.com.au Tel: +886 2 8660 9496 Fax: +886 2 8660 9342 Norway Email: [email protected] shi.co.jp Nordic BioSite AS Web: www.biopioneer.com.tw o.jp Tel: 800 103 01 Fax: +46 8756 9490 Hycell International Co., Ltd. Email: [email protected] Tel: +886 2 2945 3479 Web: www.biosite.no 0 Fax: +886 2 2945 3470 Email: [email protected] co.jp Web: www.hycell.com.tw o.jp

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TREVIGEN INC. 8405 Helgerman Court Gaithersburg, MD 20877 [email protected] 1.800. TREVIGEN Tel: 301.216.2800 Fax: 301.216.2801

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