Apoptosis Methods

Method Booklet 3

Apoptosis Applications & Glossary Apoptosis Applications Table of Contents & Glossary Introduction ...... 2 Annexin-V ...... 5 Caspases ...... 8 Fluorometric detection ...... 12 Colorimetric detection ...... 14 PARP ...... 18 BID ...... 21 DNA Fragmentation ...... 23 TUNEL ...... 25 Appendix: other apoptosis products ...... 34 References ...... 41 Glossary ...... 43

Technical/Customer Services 1-800-242-0607 Technical/Customer Services 1-800-242-0607 DNA fragmentation occurs when nuclear enzymes become active and destroy the normal Introduction chromatin structure of DNA. The DNA becomes fragmented in pieces of 200 bp or less Apoptosis, defined as programmed cell death, plays a very important role in many phys- in length. This fragmentation can easily be visualized by agarose gel. BioSource iological and pathological conditions such as embryo and organ development, immune International, Inc. provides a simple Quick Apoptotic Ladder Kit for determining if frag- responses, tumor development and growth. Detecting apoptotic cells or monitoring the mentation of DNA has occurred, allowing one to distinguish between apoptotic cells and cells progressing to apoptosis is an essential step in basic research and in developing normal cells. drugs that may regulate apoptosis. Breaks in the DNA can be further distinguished by use of bromodeoxyuridine triphos- Apoptosis is characterized by many biological and morphological changes; such as, phate (Br-dUTP). Br-dUTP incorporates into breaks in the DNA. One can then label the change of mitochondrial membrane potential, activation of caspases, DNA fragmentation, broken DNA with an antibody to Br-dUTP as provided in the APO-BRDU TUNEL kit to membrane blebbing and formation of apoptotic bodies. Based on these changes, various distinguish apoptotic cells from normal and necrotic cells by flow cytometry. assays are designed to detect or quantitate apoptotic cells. Typical assays include Annexin- A wide variety of additional reagents for apoptosis research are described in the appen- V binding, caspase enzyme activity, TUNEL (terminal deoxynucleotidyl transferase-medi- dix including antibodies, ELISA kits, mRNA kits, primers, substrates and inhibitors. ated dUTP nick-end-labeling) and DNA gel electrophoresis (see Table 1, next page). Table 1: Comparison of Annexin-V assay with Caspase-3 and TUNEL assay The goal of this manual is to provide customers with proven methods that highlight the menu of products offered by BioSource International, Inc. Our Research and Assays Annexin-V binding assay Caspase-3 assay TUNEL assay Development staff has detailed the protocols used to obtain the results given here. When Mechanism Apoptotic cells have PS DEVD-AFC (fluorometric) or Exogenous TdT incorpo- appropriate, buffer formulations and specific parameters for instruments are also provid- exposure. Annexin-V DEVD-pNA (colorimetric) rates dUTP to the ends of ed. As always, your feedback has contributed to the initiative for this publication and will conjugates bind to PS on is used as substrate to detect DNA fragments, which continue to affect improvements in the materials which are supplied with our products. the cell surface and Caspase-3 and Caspase-3 like can be detected with Please contact our Technical Service staff with any inquiries regarding this manual. identify apoptotic cells. enzyme activity. various methods. ([email protected] or 1-800-242-0607.) Stage of apoptosis Relatively early, but Early. Late. after caspase activation. The first method which is described in this publication is the detection of Annexin V-FITC by flow cytometry. This method has become a hallmark for the separation of necrotic Adherent cells or Useful for suspension cells Useful for adherent cells and Useful for adherent cells or suspension cells and difficult to perfrom on suspension cells. suspension cells. cells from those undergoing true apoptosis. BioSource International, Inc. has formulated adherent cells. a kit containing Annexin V-FITC and propidium iodide, along with detailed instructions on how to distinguish cells in early apoptosis, late apoptosis, and necrosis, utilizing a Tissues Not recommended. Not recommended. Can be used on tissue two- color staining regime. sections. Sample storage Assay done immediately Treated samples may be stored Treated cells may be fixed Quantitation of caspase activity is another popular means of determining what stage of following the induction at –20°C for further assay. and stored. apoptosis may be occurring. Caspases are protein-cleaving enzymes which are instru- of apoptosis. mental in the sequential disassembly of cells. BioSource International, Inc. has formulat- Multiple color Can be used for multiple May not be used with other Potential multiple color ed kits for screening cell lysates for Caspase-2, -3, -6, -8, and -9 activity by either fluoro- analysis color analysis and markers. analysis. Analyzes metric or colorimetric detection. Also available are antibodies for Caspase-1, -3, -6, -8 analyzing apoptosis at single apoptotic cells at single and -10 for use in Western blotting. cell level. cell level.

A novel method for detection of apoptosis by flow cytometry is the PARP-FITC Cleavage Time consuming Simple procedure and short Several procedures and Multiple procedures and and procedures incubation, 15 minutes. long incubation (1-2 hours). multiple washing needed. Site-Specific Antibody (CSSA). This FITC-conjugated anti-PARP antibody specifically recognizes the 85kDa fragment of cleaved PARP which can be an excellent marker for detect- Number of samples More samples (decades) Many samples (hundreds) Limited samples can be ing apoptosis. Along with the PARP-FITC CSSA, BioSource International, Inc. also provides can be handled can be done in one can be done in one done in one experiment. an unconjugated and Biotin form of the antibody, and has formulated a PARP-FITC CSSA experiment. experiment. Kit which includes fixation and permeabilization buffers and matching peptide. Instrument Flow cytometer or Spectrofluorometer or spectro- Flow cytometer, fluor- fluorescence microscope. photometer (microtiter plate escence or light microscope. Cleaved BID (BH3 interacting domain death agonist) plays an important role in the trans- reader). duction and amplification of apoptotic signals during apoptosis. BioSource International, Non-specific Potentially high non- Some background on non- Low background on non- Inc. offers four BID antibodies that detect full length BID in both human and mouse, a background specific background if the apoptotic cells (untreated). apoptotic cells. BID p15 fragment that recognizes human BID, and a BID [59/60] CSSA that specifically cell culture condition is not recognizes cleaved mouse BID. optimal because of dead cells. e-mail: [email protected] Technical/Customer Services 1-800-242-0607 We welcome your feedback on the methods described herein. Please contact us with your comments. All methods and products are for research purpose only and not intend- Chapter 1: Annexin-V ed for diagnostic use. Introduction Disclaimer: The procedures presented in this manual are accurate to the best of our knowledge. The Annexin-V binding assay is based on the relocation of phosphatidylserine to the outer BioSource International, Inc. makes no warranties either expressed or implied as to any cell membrane. Viable cells maintain an asymmetric distribution of different phospho- matter whatsoever, including, without limitations, the condition of the products, their lipids between the inner and outer leaflets of the plasma membrane. Choline-containing merchantability, or fitness for any particular use. BioSource International, Inc., shall not phospholipids such as phosphatidylcholine and sphingomyelin are primarily located on be held liable for any direct, indirect, incidental, or consequential damages, including the outer leaflet of viable cells and aminophospholipids such as phos- without limitation, loss of profit, loss of business, or other loss which may be based direct- phatidylethanolamine and phosphatidylserine (PS) are found at the cytoplasmic (inner) ly or indirectly upon the sale, use of products, or inadequacy of product for any purpose face of viable cells. The distribution of phospholipids in the plasma membrane changes or by any defect or deficiency therein, even if BioSource International, Inc., knew or during apoptosis. In particular, PS relocates from the cytoplasmic face to the outer leaflet should have known of the possibility of such loss. so called PS exposure. The extent of PS exposure can distinguish apoptotic cells from the non-apoptotic cells. Annexin-V is a 35-36 kDa calcium-dependent phospholipid binding protein with high affinity for PS (kDa ~ 5x10-10 M). When labeled with a fluorescent The products listed in this booklet are for research use only, and dye, Annexin-V can be used as a sensitive probe for PS exposure on the outer leaflet of are not intended for use in diagnostic procedures. the cell membrane.

The binding of Annexin-V conjugates such as Annexin-V FITC to cells permits differentiation of apoptotic cells (Annexin-V positive) from non-apoptotic cells (Annexin-V negative) (see Figure 1, page 6). Annexin-V binding is observed under two conditions. The first condition is observed in cells midway through the apoptosis pathway. Phosphatidylserine translocates to the outer leaflet of the cell membrane. The second condition is observed in very late apoptosis or when the cells become necrotic and membrane permeabilization occurs. This membrane permeabilization allows Annexin-V to enter cells and bind to phosphatidylserine on the cytoplasmic face of the membrane. Since other causes besides apoptosis can result in necrosis, it is important to distinguish between necrotic and apoptotic cells. Membrane permeabilization also permits entry of other materials to the interior of the cell, including the fluorescent DNA-binding dye propidium iodide. Utilizing dual staining methodology, apoptotic populations can be distinguished from necrotic populations. For example, using the Annexin V-propidium iodide (PI) double staining regime, three populations of cells are distinguishable in two- color flow cytometry (Figure 1).

Non-apoptotic cells: Annexin-V negative and PI negative; Early apoptotic cells: Annexin-V positive and PI negative; Necrotic cells or late apoptotic cells: Annexin-V positive and PI positive.

In summary, Annexin-V FITC/PI dual staining is very useful for defining apoptotic suspension cells by flow cytometry.

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Typical Data Using Annexin-V FITC Kit 2. Dilute 10x Annexin-V Binding Buffer 1:10 in distilled water. 3. When apoptotic induction of cells is complete, wash the cells with PBS twice and A B resuspend cells at 2-3 x 106 cells/mL in 1x Annexin-V Binding Buffer. 4. Aliquot cells at 100 µL/tube. 5. Add 5 µL of Annexin-V FITC and 10 µL of Propidium Iodide Buffer to each tube. 6. Incubate at room temperature for 15 minutes in the dark. 7. Add 400 µL 1x Annexin-V Binding Buffer to each tube. 8. Analyze the cells by flow cytometry within 1 hour of staining.

Annexin-V Troubleshooting Guide Propidium Iodide Propidium

Problems Causes Solutions High background on 1. Cell culture condition 1. Change cell culture condition, use Annexin-V FITC Annexin-V FITC non-stimulated cells may be not optimal. lower cell density.

Log Green Fluorescence 2. Cells have been stored too 2. Perform staining immediately after long after the treatment. collecting cells. Figure 1: Jurkat cells were cultured with 2 µM camptothecin for 5 hours and stained by 3. Cells have high constitutive 3. Use a lower amount of Annexin-V Annexin-V FITC (Catalog #PHN1008). Apoptotic and non-apoptotic cells can be differ- PS on cell surface. conjugates per test. entiated according to Annexin-V FITC binding (A). M1 represents non-apoptotic cells and M2 represents Annexin-V positive cells. Staining with propidium iodide (Catalog Low percentage of 1. Inducers are not effective. 1. Use camptothecin to treat Jurkat #PNN1011) differentiates the Annexin-V positive population (apoptosis) from cells in apoptotic cells cells as positive control to check necrosis (B). Conclusion: propidium iodide stained cells are easily distinguished from Annexin-V conjugates and then use early apoptotic population (Annexin-V positive and PI-negative) appearing in the lower optimal induction conditions. right quadrant of the two color dot plot. 2. Cell culture condition may 2. Grow cells in optimal condition. Method not be optimal, e.g., overgrown cells may be less sensitive to stimuli. Purpose: The ApoTarget™ Annexin-V FITC Apoptosis Kit is designed to detect apoptotic cells by flow cytometry or immunofluorescence. 3. Annexin-V Binding Buffer 3. Use freshly prepared Annexin-V is not effective. Binding Buffer. The ApoTarget™ Annexin-V FITC Apoptosis Kit (Catalog #PHN1010) employs a fluorescein-labeled Annexin-V (Annexin-V FITC) in concert with propidium iodide to detect the 4. Analyzed stained cells too 4. Analyze cells within one hour cells undergoing apoptosis. long after staining. after staining.

Reagents and Equipment There is an intermediate 1. PI concentration is too 1. Reduce the concentration of 1. Annexin-V FITC (Catalog #PHN1008) PI stained population high for the cells. Propidium Iodide. 2. Propidium Iodide Buffer (Catalog #PNN1011) 3. Annexin-V Binding Buffer (Catalog #PNN1001) 4. Fluorescence activated cell sorter with a laser using excitation at 488 nm (for detec- Product Catalog # Size tion by flow cytometry) Annexin-V FITC Kit PHN1010 20 Tests 5. PBS Annexin-V FITC Kit PHN1018 300 Tests 6. Calibrated adjustable precision pipettes, preferably with disposable plastic tips Annexin-V Biotin PHN1009 100 Tests 7. Tubes appropriate for holding cells during induction of apoptosis Annexin-V Cy3 PHN1000 100 Tests 8. Microcentrifuge Annexin-V FITC PHN1008 100 Tests Annexin-V Binding Buffer PNN1001 50 mL Assay Procedure Propidium Iodide Buffer PNN1011 2 mL 1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Through the use of synthetic peptides, Caspases have been divided into three groups based Chapter 2: Caspases on the four amino acids amino-terminal to their cleavage site. Caspases-1, -4 and -5 prefer substrates containing the sequence WEXD (where X is variable). Caspases-2, -3 and - Introduction 7 prefer the sequence DEXD. Caspases 6, 8 and 9 are the least demanding but have demonstrated a preference for cleaving of substrates containing either LEXD or VEXD. One group of cysteinyl-aspartic acid proteases is called caspases. Caspases are members Because these sequences correspond to known cleavage sites of caspase targets, systems of the Interleukin-1β Converting Enzyme (ICE) family of cysteine proteases. There are 14 to study caspase cleavage activity have been developed. The measurement of caspase caspases that have been identified which include: enzyme activity with fluorometric and colorimetric peptide substrates and the detection of caspase cleavage using antibodies to caspases allows the study of the apoptosis processes Caspase-1 Also known as ICE or screening of therapeutic agents which promote or prevent apoptosis. BioSource Caspase-2 Also known as Ich-1, Nedd2 International, Inc. provides Caspase-2, -3, -6, -8 and -9 protease activity assay kits (both Caspase-3 Also known as CPP32, Yama, Apopain, SCA-1, and LICE fluorometric and colorimetric) as well as antibodies specific for Caspase-3,-6,-8 and -10. Caspase-4 Also known as ICEreI-II, TX and ICH-2 Caspase-5 Also known as ICErel-III and TY Caspase-6 Also known as Mch-2 Caspase-7 Also known as Mch-3, ICE-LAP-3 and CMH-1 Figure 1: Caspase-8 Also known as FLICE, Mach-1 and Mch5 Caspase-9 Also known as ICE-LAP6, Mch6 and Apaf-3 Caspase-10 Also known as FLICE-2, Mch4 Caspase-11 Caspase-12 Caspase-13 Also known as ERICE Caspase-14 Also known as Mini-ICE

Phylogenetic analysis of caspases reveals that there are four subfamilies:

Subfamily Caspase ICE subfamily Caspase-1, -4 and –5 Ced-3/CCP32 subfamily Caspase-3,-6, -7 Mach/FLICE subfamily Caspase-8, and –10 ICH (ICE and CED-3 homologue) subfamily Caspase-2 and -9

The caspases are synthesized as inactive pro-enzymes or pro-caspases. In apoptosis, the pro-caspases are processed by proteolytic cleavage to form active enzymes. For example, Caspase-3 exists in cells as an inactive 32 kDa proenzyme, called pro-Caspase-3. Pro- Caspase-3 is cleaved into active 17 and 12 kDa subunits by upstream proteases to become active Caspase-3. Caspases-2, -8, -9 and -10 are classified as signaling or “upstream” (see Figure 1, next page) in the apoptosis pathway because long prodomains allow association with cell surface receptors such as FAS (CD95), TNFR-1 (CD120a), DR- 3 or CARD domains . This observation suggests a proteolytic cascade as a mechanism for signaling. A proteolytic cascade exists that would activate the terminal event required for apoptosis in a way similar to that of the coagulation cascade seen with the closely related family of serine proteases. For example, Caspase-4 activates pro-Caspase-1; Caspase- 9 activates pro-Caspase-3; and Caspase-3 cleaves pro-Caspase-6 and pro-Caspase-7.

Caspases play a critical role in the execution phase of apoptosis. Important targets of caspases include cytoplasmic and nuclear proteins such as keratin 18, poly ADP ribose polymerase (PARP) and lamins. Overexpression of Caspase-3 induces apoptosis. e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Methods for Detecting Caspase Activity amino acid sequence of DEVD. The peptide substrate used would be DEVD with the D Figure 2: labeled with a colorimetric or fluorometric marker. The marker is then allowed to fluo- The excitation and emission wavelengths for the fluorochromes of resce or emit color once cleavage has occurred. Without cleavage, little or no signal caspase substrates available from BioSource International, Inc. should be observed. Thus, utilizing DEVD-AFC (Catalog #77-934), DEVD-AMC (Catalog Excitation Emission #77-910) and MCA-DEVDARK[K-DNP] (Fluorescence Resonance Energy Transfer (FRET) peptide) (Catalog #77-966), cleavage of the peptide by the caspase can be observed. Figure 2 provides the information on the excitation and emission wavelengths for those fluorochromes of those caspase subsrates. See page 18 for a complete listing of caspase kits.

AFC Purpose: Following are two examples for in vitro determination of Caspase-3 proteolytic activity in lysates of mammalian cells using our simple and convenient caspase activity assay kits. Both methods provide a means for quantitating caspases that recognize the amino acid sequence, DEVD. One utilizes the fluorophore, AFC, for fluorometric analysis of samples, and the other is a colorimetric method utilizing pNA as a marker. The kits include substrate and optimized buffers for the appropriate dye.

300 350 400 450 500 550 600 350 400 450 500 550 600 Caspase Protease Activity Assay

Induction of apoptosis in cells

AMC

Protease activation

300 350 400 450 500 550 600 350 400 450 500 550 600

DEVD-AFC DEVD-pNA

FRET Caspase-3 (CPP32)

DEVD DEVD

300 350 400 450 500 550 600 350 400 450 500 550 600

Wavelength (nm) AFC pNA Caspase proteins cleave other proteins after aspartic acid. From previous studies we know that the three to four amino acids prior to the aspartic acid confer specificity to the caspase. This allows the use of four amino acid-labeled peptides to be utilized as substrates for the caspases. For example, the clevage site for Caspase-3 corresponds to the e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Fluorometric Method: Caspase-3 Assay Procedure 1. Induce apoptosis in cells by desired method. Concurrently incubate a control cul- The substrate, DEVD-AFC, is composed of the fluorophore, AFC (7-amino-4-trifluo- ture without induction. romethyl coumarin), and a synthetic tetrapeptide, DEVD (Asp-Glu-Val-Asp), which is the 2. When the induction is complete, count cells and pellet 1-5 x 106 cells or use 20- upstream amino acid sequence of the Caspase-3 cleavage site in PARP. DEVD-AFC emits 200 µg cell lysate, if protein concentration has been determined. blue light (λ max = 400 nm). Upon cleavage of the substrate by Caspase-3 or related cas- 3. Resuspend cells in 50 µL of chilled Cell Lysis Buffer and incubate cells on ice for 10 pases, with the excitation wavelength set to 400 nm, free AFC emits a yellow-green flu- minutes. orescence (λ max = 505 nm) which can be quantified using a spectrofluorometer or a flu- 4. Prepare reaction buffer: orescence microtiter plate reader. Comparison of the fluorescence of AFC from apoptot- Number of Samples Amount of 2x Reaction Buffer Amount DTT ic samples with an uninduced control allows determination of the increase in Caspase-3 (sample x 50 µL) (10 µL x 2x vol.) activity. 5 250 µL 2.5 µL 10 500 µL 5 µL Reagents and Materials 25 1.250 mL 12.5 µL 1. Cell Lysis Buffer Determine the number of samples to be measured and aliquot enough 2x Reaction 2. 2x Reaction Buffer Buffer into a glass tube (assuming 50 µL of 2x Reaction Buffer per sample). Add DTT 3. Substrate DEVD conjugated to the fluorophore AFC (Catalog #77-934) to the 2x Reaction Buffer immediately before use (10 mM final concentration: add 4. 1M DTT 10 µL of 1.0 M DTT stock per 1 mL of 2x Reaction Buffer). 5. Fluorometer (and cuvettes) or fluorescent microplate reader equipped with excita- 5. Add 50 µL of 2x Reaction Buffer (containing 10 mM DTT) to each sample. tion filter 370-425 nm and emission filter 490–530 nm. (maximal ex = 400 nm and 6. Add 5 µL of the 1 mM DEVD-AFC substrate (50 µM final concentration) and incu- em = 505 nm) (See figure 2) bate at 37°C for 1-2 hours. Keep the samples in the dark during incubation. 6. Calibrated adjustable precision pipettes, preferably with disposable plastic tips 7. Read sample in a fluorometer or fluorescent microplate reader with a 400 nm exci- 7. Protein measurement method, such as Bradford protein assay tation filter and 505 nm emission filter. 8. Tubes appropriate for holding cells during induction of apoptosis 8. Fold-increase in Caspase-3 activity should be determined by direct comparison to 9. Microcentrifuge the level of the uninduced control. 10. Reaction tubes or 96-well microplate Typical Data Using Fluorescent Caspase-3 Activity Assay Kit (Cat.# KHZ0012) Procedure Summary for Caspase Assays (Fluorometric) The following absorbance data were obtained from Jurkat cells. Apoptosis was induced by incubating Jurkat cells with 0.2 µg/mL of anti-Fas monoclonal antibody (Cat.# Collect cells and resuspend AHS9552) for 8 hours. The assay was performed according to the procedure described in Lysis Buffer above. Figure 3: 10 minutes on ice

Add Reaction Buffer containing DTT 900 800 700 Add Conjugated Protease 600 Substrate 500 400 300 37°C for 1 hour 200 Analyze using a 100 Fluorometer 20 40 80 120 160 200 30 λ ex=400 nm, λ em=505 nm

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Caspase Fluorometric Assay Kit Troubleshooting Guide 8. Protein measurement method, such as Bradford protein assay 9. Tubes appropriate for holding cells during induction of apoptosis Problems Causes Solutions 10. Microcentrifuge Unexpected high fluo- 1. Gain of fluorescence reader 1. Decrease gain. 11. Reaction tubes or 96-well microplate rescence development is too high. Procedure Summary for Caspase Assays (Colorimetric) 2. Too much protein. 2. Use less protein or dilute sample.

Weak or no signal 1. Incubation of substrate in 1. Only use substrate solution contain- Collect cells and resuspend incubation buffer without DTT. ing DTT. in Lysis Buffer

2. The caspase is digested 2. Perform lysis of cells on ice and 10 minutes on ice nonspecifically. store at –20°C.

3. Induction of apoptosis is 3. Check inducers and conditions. Centrifuge and collect not successful. Supernatant

4. Unsuitable filters have 4. Check the filters for correct wave- been used. length. Add Reaction Buffer containing DTT 5. Gain of fluorescence reader 5. Increase gain. is too low.

Poor precision 1. Non-homogeneous sample 1. Mix sample well before pipetting. Add Conjugated after freezing. Protease Substrate

2. Turbidity, particle or high 2. Centrifuge sample. 37°C for 1 hour lipid or DNA content within the sample. Analyze using a Spectrophotometer 3. Unequal volumes added 3. Check pipette function. At 405 nm to wells.

Colorimetric Method: Caspase-3 Assay Procedure The substrate, DEVD-pNA, is composed of the chromophore, p-nitroanilide (pNA), and a 1. Induce apoptosis in cells by desired method. Concurrently incubate a control synthetic tetrapeptide, DEVD (Asp-Glu-Val-Asp), which is the upstream amino acid culture without induction. sequence of the Caspase-3 cleavage site in PARP. Upon cleavage of the substrate by 2. When induction is complete, count cells and pellet 3-5 x 106 cells per sample. Caspase-3 or related caspases, free pNA light absorbance can be quantified using a spec- 3. Resuspend cells in 50 µL of chilled Cell Lysis Buffer and incubate cells on ice trophotometer or a microplate reader at 400 or 405 nm. Comparison of the absorbance for 10 minutes. of pNA from apoptotic sample with an uninduced control allows determination of the 4. Centrifuge for 1 minute in a microcentrifuge (10,000 x g). increase in Caspase-3 activity. 5. Transfer supernatant (cytosol extract) to a fresh tube and put on ice. Reagents and Materials 6. Assay protein concentration by any standard method. 1. Cell Lysis Buffer 7. Dilute each cytosol extract to a concentration of 50-200 µg protein per 50 µL 2. 2x Reaction Buffer Cell Lysis Buffer (1-4 mg/mL). 3. Substrate DEVD conjugated to the chromophore, pNA (Catalog #77-900) 8. Prepare Reaction Buffer: 4. Dilution Buffer Determine the number of samples to be measured and aliquot enough 2x Reaction 5. 1M DTT Number of Samples Amount of 2x Reaction Buffer Amount DTT 6. Spectrophotometer (and cuvettes) or microplate reader capable of measurement (sample x 50 µL) (10 µL x 2x vol.) at 400-405 nm 5 250 µL 2.5 µL 7. Calibrated adjustable precision pipettes, preferably with disposable plastic tips 10 500 µL 5 µL 25 1.250 mL 12.5 µL e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Buffer into a glass tube (assuming 50 µL of 2x Reaction Buffer per sample). Add DTT Caspase Colorimetric Assay Kit Troubleshooting Guide to the 2x Reaction Buffer immediately before use (10 mM final concentration: add 10 µL of 1.0 M DTT stock per 1 mL of 2x Reaction Buffer). Problems Causes Solutions 9. Add 50 µL of 2x Reaction Buffer (containing 10 mM DTT) to each sample. Weak or no signal 1. Incubation of substrate in 1. Only use substrate solution contain- 10. Add 5 µL of the 4 mM DEVD-pNA substrate (200 µM final concentration) and incu- incubation buffer without DTT. ing DTT. 2. The caspase is digested 2. Perform lysis of cells on ice and bate at 37°C for 2 hours. Keep the samples in the dark during incubation. nonspecifically. store at –20°C. 11. Read samples at 400 nm or 405 nm in a microplate reader, or spectrophotometer 3. Induction of apoptosis is 3. Check inducers and conditions. using a 100 µL micro-quartz cuvette, or dilute sample to 1 mL with Dilution Buffer not successful. and use a regular cuvette. 4. Protein concentration is 4. Increase protein concentration. too low. Note: Dilution of the samples proportionally decreases the optical density. You may also 5. Inadequate incubation time 5. Increase incubation time. perform the entire assay directly in a 96-well plate. or temperature. High background on 1. Cell culture condition is 1. Check cell culture condition. non-apoptotic cells not optimal. 12. Increase in Caspase-3 activity should be determined by direct comparison to the Poor precision 1. Non-homogeneous sample 1. Mix sample well before pipetting. level of the uninduced control. after freezing. Uninduced cells Induced cells 2. Turbidity, particle or high 2. Centrifuge sample. (non apoptotic) (apoptotic) lipid or DNA content within Lysate (50 µL) ++++ the sample. 2x Reaction Buffer (50 µL) ++++ 3. Unequal volumes added 3. Check pipette function. DEVD-pNA (5 µL) - + - + to wells. Control 1 Control 2 Control 3 Testing Limitations of the Procedure Calculation: Fold Increase = (Testing OD-Control 3 OD)/(Control 2 OD-Control 1 OD) These kits provide a simple and convenient method to detect Caspase-3 activity of apoptotic cells. For fluorescent kits, if the reading of relative fluorescence is off scale high, the Note: Background readings from cell lysates and buffers should be subtracted from the samples can be proportionally diluted with PBS. For both fluorescent and colorimetric readings of both induced and uninduced samples before calculating fold-increase in assays, a relatively high concentration of DTT (10 mM) is required for full activity of the Caspase-3 activity. caspases. Make sure that DTT is added to the Reaction Buffer when the assay is carried out; otherwise, unexpected low caspase activity will occur. Turbidity, lipemia or particu- Typical Data Using Colorimetric Caspase-3 Activity Assay Kit (Cat.# KHZ0022) late materials in samples can decrease the assay precision. The following absorbance data were obtained from Jurkat cells. Apoptosis was induced * Also available as 50 Test size. Please inquire for catalog number and pricing. by incubating Jurkat cells with 0.2 µg/mL of anti-Fas monoclonal antibody (Cat.# Product Catalog # Size AHS9552) for 8 hours. The assay was done according to the procedure described above. Caspase-2 Colorimetric Assay* KHZ0082 200 Tests Typically, the Caspase-3 activity in 20 µg-200 µg protein per sample can be detected. Caspase-3 Fluorometric Assay* KHZ0012 200 Tests Figure 4: Caspase-3 Colorimetric Assay* KHZ0022 200 Tests Caspase-6 Fluorometric Assay* KHZ0032 200 Tests Caspase-6 Colorimetric Assay* KHZ0042 200 Tests Caspase-8 Fluorometric Assay* KHZ0052 200 Tests Caspase-8 Colorimetric Assay* KHZ0062 200 Tests Caspase-9 Colorimetric Assay* KHZ0102 200 Tests Caspases -2, -3, -6, -8, -9 Colorimetric Assay Sampler Kit KHZ1001 125 Tests Anti-caspase-1/ICE (human, mouse, rat) AHZ0082 100 µg Anti-caspase-3 (human, mouse, rat) AHZ0052 100 µg Anti-caspase-6 (human) AHZ0062 100 µg Anti-caspase-8 (human), (Clone 5F7) AHZ0072 100 µg Anti-caspase-8 (human), (Clone 12F5) AHZ0502 100 µg Anti-caspase-10 (human) AHZ0092 100 µg Recombinant Human Caspase-3/CCP32 PHZ0014 100 Units Recombinant Human Caspase-6 PHZ0034 100 Units e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Chapter 3:PARP

Introduction 2% 52% 34% 22% Protein cleavage, or proteolysis, is a general process in the cells and is involved in many cell activities including the activation of the pro-enzymes, production of functional proteins, and cell signaling. For example, under normal conditions, caspases are present in cells as inactive proenzymes, or procaspases. Upon induction of apoptosis, procaspases are activated through proteolytic processing. Active caspases lead to a self-amplifying cascade of proteolysis and cleavage of many cellular proteins including PARP (Poly (ADP- Staurosporine dose-response HeLa cells. From left to right, untreated, 1.0, 0.5 and 0.25 µM staurosporine. M2 rep- Ribose) Polymerase). PARP is a 116 kDa nuclear protein which is strongly activated by resents the percentage of apoptotic cells in each sample. DNA strand breaks. PARP plays a role in DNA repair as well as in other cellular processes, including DNA replication, cell proliferation and differentiation. During apoptosis, Protocol for Staining Suspension Cells by Flow Cytometry ICE family members, such as caspase-3 and -7, cleave PARP to yield an 85 kDa and a 25 1. Treat Jurkat cells with 0.25, 0.125 and 0.06 µg/mL of anti-Fas mAb (Cat.# AHS9552) for 1 hour. Use untreated Jurkat cells as a control. kDa fragment. PARP cleavage is considered to be one of the classical characteristics of 2. Collect cells and wash 2 x in PBS. apoptosis. BioSource International, Inc. offers an anti-PARP-FITC conjugated Cleavage 3. Fix cells in IC Fix™ buffer (Cat. # FB001) containing 4% paraformaldehyde for 20 minutes at 4°C. Site-Specific Antibody (CSSA) (Cat. #44-699) that can detect apoptotic cells by flow 4. Permeablize cells in IC Perm™buffer (Cat. # PB001) for 10 minutes. cytometry. An alternative to the TUNEL assay, the PARP-FITC CSSA can detect apoptosis 5. Add 10 µL PARP-FITC CSSA (Cat. # 44-699) to 105 cells in 100 µL IC Perm™ buffer and incubate for in adherent and suspension cells. The PARP-FITC CSSA is also available in kit form (Cat. 30 minutes at RT. #AHM2011). An unconjugated PARP CSSA (Cat. #44-698) and a Biotin conjugated form 6. Wash the cells 2 x in IC Perm™buffer. (Cat. #44-697) can be used for other applications. 7. Resuspend cells in PBS and analyze cells by FACS.

Detection Flow Cytometry IHC WB Methods Adherent Suspension PARP CSSA + + + + TUNEL + + + - Annexin-V +/- + - -

Protocol for Staining Adherent Cells by Flow Cytometry 1. Treat HeLa cells with 1, 0.5 and 0.25 µM staurosporine for 5 hours. Induction of apoptosis in Jurkat cells by anti-Fas mAb. From left to right, untreated, 0.25, 0.125, and 0.06 µg/mL anti-Fas mAb. M2 represents the percentage of apoptotic cells in each sample. Use untreated HeLa as a control. 2. Keep culture supernatant (some apoptotic cells are detached). 3. Wash cells 2 x in situ with PBS. Protocol for Immunoblotting 4. Add 2 mM EDTA to the cells and check under a microscope. 1. Perform SDS-PAGE on cell lysates treated with apopotsis inducers, or untreated cells (as a control) 5. Once detached, collect cells and add to culture supernatant. and transfer the protein onto PVDF membrane. 6. Wash cells 1 x with PBS. 2. Block the membrane in Blocking Buffer (5% non-fat milk in tTBS) for 30 minutes at RT. 7. Fix cells in IC Fix™ buffer (Cat. # FB001) containing 4% paraformaldehyde 3. Incubate the membrane in 0.5 µg/mL of anti-PARP CSSA (Cat. # 44-698) in Blocking Buffer for 1 for 20 minutes at 4°C. hour at RT. 8. Permeablize cells in IC Perm™ buffer (Cat.# PB001) for 10 minutes. 4. Wash the membrane 3 x with Washing Buffer (tTBS). 9. Add 10 µL PARP-FITC CSSA (Cat. # 44-699) to 10 5. Incubate the membrane with goat F(ab’) anti-rabbit IgG alkaline phosphatase (Cat.# ALI4405) at 5 cells in 100 µL IC Perm™ buffer and incubate for 2 30 minutes at RT. 1:5000 dilution for 1 hour at RT. 10. Wash cells in IC Perm™ buffer. 6. Wash the membrane 3 x with Washing Buffer. 11. Resuspend cells in PBS and analyze cells by FACS. 7. Develop in Tropix WesternStar™ buffer for 5 minutes and expose to film.

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Chapter 4: BID

BID,known as BH3 interacting domain death agonist, is an intracellular amplifier of apoptotic signals and is a “BH3 domain only” protein of the Bcl-2 family. Cleavage of BID plays an important role in the transduction and amplification of apoptotic signals during apoptosis. Typically, there are two major known apoptotic signal pathways during apoptosis: 1) The caspase-8 pathway initiated by stimulation of Fas or TNF-α (death receptor Detection of PARP cleavage in HeLa cells by Western blotting. agonists) and 2) The Caspase-9/cytochrome c pathway induced by irradiation or cytotoxic drugs. Cleaved BID serves as a direct link between the caspase-8 pathway and caspase- Protocol for Immunocytochemistry 9/cytochrome c death machinery. 1. Grow HeLa cells on chamber slide and treat with staurosporine. Use untreated cells as a control. 2. Discard culture supernatant and fix cells in cold acetone for 2 minutes. BID usually exists as a 22 kDa inactive protein in the cytosol of living cells. It becomes α 3. Wash cells in PBS. cleaved and activated by caspase-8 in response to apoptotic stimuli such as TNF- , Fas 4. Block endogenous peroxidase by incubating cells on slides with 0.03 M and TRAIL. The cleavage of BID by caspase-8 occurs at the amino acid residues NaN and 0.03% HO in PBS for 10 minutes and rinse slide 3 x in PBS. Asp59/Gly60 in mouse and Asp60/Gly61 in human. The C-terminal part of cleaved BID 3 2 2 [p15] translocates onto mitochondria and triggers cytochrome c release. 5. Block with Blocking Buffer (5% BSA in PBS) for 30 minutes at RT. 6. Apply anti-PARP CSSA (cat. # 44-698) at 0.5 µg/mL in Blocking Buffer to slide and incubate it for 1 hour at RT and rinse slide 3 x in PBS. BioSource has 4 BID antibodies as part of our apoptosis product line. Two of these anti- 7. Apply biotinylated goat F(ab’) bodies recognize human BID; one recognizes only full length human BID, the other rec- 2 anti-rabbit IgG (Cat. # ALI4409) at 1: 200 dilution in Blocking Buffer for 1 hour at RT and rinse slide 3 x in PBS. ognizes the p15 portion of BID as well as full-length human BID (see figure below for 8. Apply ABC reagent (Vectastain Elite) for 30 minutes and rinse slide 3 x in PBS. data). The other two antibodies specifically recognize mouse BID; one recognizes only 9. Add DAB substrate for 5 minutes and wash the slide 3 x in dHO. full length mouse BID, the other is a cleavage site-specific antibody (CSSA) to mouse BID 2 [59/60]. The CSSA to mouse BID [59/60] specifically recognizes the 15 kDa cleaved BID 10. Counterstain, dehydrate, and seal the slide with coverslip. fragment.

Staurosporine treated HeLa cells

Full length BID

Cleaved BID, p15 Untreated

1 2 3 4

PARP CSSA stained apoptotic HeLa cells treated with staurosporine. Proteins were resolved from recombinant human BID (Lane 1), caspase-8 treated human BID (Lane 2), Jurkat cells (Lane 3), and the apoptotic Jurkat cells induced by anti-Fas antibody (Cat. #AHS9552) (Lane 4) using SDS-PAGE. The proteins were incubated with the anti-human BID (p15) antibody at 0.5 µg/mL.

Product Catalog # Size The data show that the anti-human BID (p15) antibody recognizes the 15 kDa BID fragment cleaved by caspase-8 and PARP [214/215] FITC Conjugated Kit AHM2011 100 tests in apoptotic Jurkat cells (Lane 2 and 4). PARP [214/215] FITC CSSA 44-699 100 tests PARP [214/215] CSSA Unconjugated 44-698 100 µg PARP [214/215] CSSA Biotin 44-697 100 tests

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Chapter 5: DNA Fragmentation

Purpose: The ApoTarget™ Quick Apoptotic DNA Ladder Detection Kit is designed for preparation of nucleic acids from mammalian cells to determine the level of DNA frag- Full length BID mentation of apoptotic cells. Cleaved BID Principle of the Method 1 2 3 4 5 1 2 3 4 5 Internucleosomal DNA fragmentation is considered a hallmark of apoptosis. During apoptosis, activated nucleases degrade the higher order chromatin structure of DNA into fragments of 50 to 300 kilobases and subsequently into small DNA pieces of about 200 base Mouse L929 cells were treated with 5 µg /mL actinomycin plus 2.5 ng/mL, 5 ng/mL and 10 ng/mL mouse TNF-alpha (Cat. pairs in length. These DNA fragments can be extracted from cells and visualized by hori- # PMC3014) for 5 hours (Lane 3, 4 and 5). Untreated L929 cells (Lane 1) and actinomycin treated L929 cells (Lane 2) zontal gel electrophoresis followed by ethidium bromide staining. The detection of DNA were used as controls. Cells were incubated with the anti-mouse full length BID antibody (Cat. #44-434, Left panel) and anti-mouse BID CSSA [59/60] (Cat. #44-436, Right panel). fragments by gel electrophoresis is one method to identify cells undergoing apoptosis.

The data show that anti mouse full length BID antibody only reacts with a 22 kDa mouse full length BID (Left panel). The The ApoTarget™ Quick Apoptotic DNA Ladder Detection Kit (Catalog #KHO1021) pro- mouse BID CSSA [59/60] recognizes the cleaved mouse BID in apoptotic L929 cells (Right panel). vides a simple and rapid procedure for extraction of chromosomal DNA. The procedure prepares DNA for use in the methods that detect DNA fragmentation in apoptotic cells. Unlike other methods which require 1 to 2 days to finish, this detection method only requires less than 90 minutes to prepare DNA in a single tube, without the need for extractions or column steps. DNA fragmentation can be easily visualized by agarose gel electrophoresis. This procedure increases recovery of small fragmented DNA and, there- Product Catalog # Size fore, improves the sensitivity of the assay. Anti-human BID [p15] 44-433 0.1 mg Anti-human Full Length BID 44-730 0.1 mg Reagents and Materials Anti-mouse BID [59/60] 44-436 0.1 mg 1. TE Lysis Buffer Anti-mouse Full Length BID 44-434 0.1 mg 2. Enzyme A Solution 3. Enzyme B Solution 4. Ammonium Acetate Solution 5. DNA Suspension Buffer 6. Agarose and TBE Buffer (1 L TBE buffer contains 5.4 g Tris, 2.8 g Boric Acid, 2 mL of 0.5 M EDTA solution, pH 8.0) 7. PBS 8. Ethidium Bromide 9. Ethanol 10. DNA ladder marker 11. Microcentrifuge 12. DNA electrophoresis equipment 13. UV light source and camera Assay Procedure 1. Induce apoptosis in cells by desired method. Concurrently incubate a control without induction. Pellet 0.25-2.0 x 106 cells in a 1.5 mL microcentrifuge tube. 2. When the induction is complete, wash cells with PBS and pellet cells by centrifugation for 5 minutes at 500 x g. 3. Carefully discard the supernatant. 4. Lyse the cells with 20 µL TE Lysis Buffer by carefully pipetting up and down several times.

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 5. Add 5 µL Enzyme A Solution to the crude lysate. Mix by gentle vortexing and incubate at 37°C in a waterbath for 10 minutes. Chapter 6: TUNEL Detection 6. Add 5 µL Enzyme B Solution to each sample and incubate at 37°C for 30 minutes in a waterbath or until the lysate becomes clear. Introduction of TUNEL Assay 7. Add 5 µL Ammonium Acetate Solution and 100 µL of absolute ethanol (kept at-20°C) to each sample. Vortex and allow the DNA to precipitate at -20°C for 10 to 15 minutes. Cell death by apoptosis is characterized by DNA fragmentation in 200-250 and/or 30-50 8. Centrifuge the sample for 10 minutes at 12,000 to 14,000 x g to collect the precip- kilobases. Further internucleosomal DNA fragmentation in 180-200 base pairs may also itated DNA. occur. Such characteristics have been used to distinguish apoptotic cells from normal or 9. Carefully discard the supernatant. necrotic cells. To detect apoptotic cells, whatever the pattern of DNA fragmentation, the 10. Add 0.5 mL of 70% cold ethanol to wash the DNA pellet and re-centrifuge the sam- TUNEL (Terminal deoxynucleotidyl transferase (TdT) mediated dUTP Nick End Labeling) ple 10 minutes at 12,000 to 14,000 x g. method is commonly utilized. In the TUNEL assay (Apo-BrDU) exogenous TdT is used to 11. Discard the supernatant and air-dry the DNA pellet for 10 minutes at room temper- catalyze a template-independent addition of bromodeoxyuridine triphosphates (Br-dUTP) ature. to the free 3’-hydroxyl ends of double or single stranded DNA fragments. After incorpo- 12. Add 30 µL of DNA Suspension Buffer, and resuspend the DNA by carefully pipet- ration, the labeled BrDU can be identified by FITC conjugated anti-Bromodeoxyuridine ting up and down several times. (BrDU) antibodies and analyzed using a flow cytometer or a fluorescence microscope. 13. Load 15 to 30 µL of each sample onto a 1% agarose gel containing 0.5 µg/mL ethid- Due to the many free 3’-hydroxyl ends of fragmented DNA in apoptotic cells, a good sig- ium bromide in both gel and running buffer (1x TBE). nal is generated in affected cell populations. These cells can be visualized in tissue sec- 14. Run the gel at 5 V/cm for 1 to 2 hours. tions or quantified with flow cytometry. Compared to a single-step labeling FITC conju- 15. Ethidium bromide-stained DNA can be visualized by transillumination with UV light gated dUTP, this two step method provides a more sensitive, stronger signal. and photographed. Method: ApoBrDU Detection Typical Data Using DNA Ladder Detection Kit Description of Kit Figure 1: The BioSource International, Inc. APO-BRDU™ Kit is a two color staining method for labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow cytometry. The kit contains the instructions and reagents required for measuring apoptosis in cells including positive and negative control cells for assessing reagent performance; washing, reaction and rinsing buffers for processing individual steps in the assay; terminal deoxynucleotidyl tranferase enzyme (TdT), bromodeoxyuridine triphosphate (Br-dUTP), fluorescein labeled anti-BrDU antibody for labeling DNA breaks and propidium iodide/RNase A solution for counterstaining the total DNA. Apoptosis was induced in Jurkat cells by incubating cells with 2 µM Camptothecin for 6 hours at Reagents and Materials 37°C (Lane 2). Jurkat cells without Camptothecin 1. Positive Control Cells were used as control (Lane 1). Detection of DNA 2. Negative Control Cells fragmentation was performed as described above. 3. Wash Buffer 4. Reaction Buffer 5. TdT Enzyme 6. Br-dUTP 7. Rinsing Buffer 1 2 8. Fluorescein~PRB-1 mAb 9. I/RNase Staining Buffer 10. Flow Cytometer 11. Distilled water 12. 1% (w/v) paraformaldehyde (methanol free) in Phosphate Buffered Saline (PBS) Product Catalog # Size 13. 70% (v/v) ethanol Quick Apoptotic DNA Laddering Kit KHO1021 50 tests

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 14. 37° C water bath Summary Diagram of APO-BRDU™ Methodology 15. Ice bucket 16. 12 x 75 mm flow cytometry test tubes 17. Pipettes and pipetting aids

Measurable Features of Apoptosis Induce apoptosis One of the most easily measured features of apoptotic cells is the break-up of the genom- in cells ic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result in the appearance of “DNA laddering” when the DNA is analyzed by agarose gel electrophoresis. The DNA of non-apoptotic cells which remains largely intact does not display this “laddering” on agarose gels during electrophoresis. The large number of DNA fragments appearing in apoptotic cells results in a multitude of 3’-hydroxyl Fixed control cells Fix cells ends in the DNA. This property can be used to identify apoptotic cells by labeling the (see Page 24) from kit 3’-hydroxyl ends with bromolated deoxyuridine triphosphate nucleotides (Br-dUTP). The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes a template independent addition of deoxyribonucleoside triphosphates to the 3’-hydroxyl ends of double- or single-stranded DNA with either blunt, recessed or overhanging ends. A substantial number of these sites are available in apoptotic cells providing the basis for the method utilized Wash cells in the APO-BRDU™ Kit. Recent evidence has demonstrated that Br-dUTP is more read- ily incorporated into the genome of apoptotic cells than are the deoxynucleotide triphosphates complexed to larger ligands like fluorescein, biotin or digoxigenin. This greater incorporation gives rise to a stronger flow cytometry signal when the Br-dUTP sites are Label DNA in cells identified by a fluorescein labeled anti-BrdU monoclonal antibody. Non-apoptotic cells do not incorporate significant amounts of the Br-dUTP owing to the lack of exposed 3’- hydroxyl DNA ends. Rinse cells

Stain cells with Fluorescein~PRB-1

TdT Propidium Iodidie + Fluorescein PRB-1 RNAse A Br-dUTP Treatment

Flow Cytometry analysis DNA Strand Breaks Add Br-dUTP To 3’-OH DNA Antibody Labeled Ends Break Sites Figure 2: Flow diagram used in the APO-BRDU™ Apoptosis Assay. The positive and negative control cells are supplied in the kit and are already fixed. The cells supplied Figure 1: Diagrammatic representation of the addition of bromodeoxyuridine triphos- by the researcher should be fixed by the researcher according to a protocol suggested phate (Br-dUTP) catalyzed by terminal deoxynucleotidyl transferase (TdT) to the 3’-OH on next page. sites of DNA strand breaks induced in the genome of apoptotic cells.

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Cell Fixation Procedure for APO-BRDU™ PROTOCOL

APO-BRDU™ Assay The following protocol describes the method for measuring apoptosis in the positive and negative control cells that are provided in the APO-BRDU™ kit. NOTE: Cell fixation using paraformaldehyde is a required step in the APO-BRDU™ assay. The same procedure should be employed for measuring apoptosis in the cell The following cell fixation procedure is a suggested method. Variables such as cell origin and specimens provided by the researcher. growth conditions can affect the results. The fixation conditions provided below should be considered as guidelines. Additional experimentation may be required to obtain results com- 1. Resuspend the positive and negative control cells by swirling the vials. Remove 1 parable to the control cells provided with the kit. The positive and negative control cells pro- mL aliquots of the control cell suspensions (approximately 1 x 106 cells per 1 mL) vided in the APO-BRDU™ KIT are already fixed. and place in 12 x 75 mm flow cytometry centrifuge tubes. Centrifuge (300 x g) the control cell suspensions for 5 minutes and remove the 70% (v/v) ethanol by aspira- 1. Suspend 1-2 x 106 cells in 0.5 mL of 10 mM sodium phosphate pH 7.2, 150 mM sodi- tion, being careful to not disturb the cell pellet. um chloride (PBS). 2. Resuspend each tube of control cells with 1 mL of Wash Buffer for each tube. 2. Add the cell suspension into 5 mL of 1% (w/v) paraformaldehyde in PBS and place on Centrifuge as before and remove the supernatant by aspiration. ice for 15 minutes. 3. Repeat the Wash Buffer treatment (step 2). 3. Centrifuge cells for 5 minutes at 300 x g and discard the supernatant. 4. Resuspend each tube of the control cell pellets in 50 µL of the DNA Labeling 4. Wash the cells in 5 mL of PBS then pellet the cells by centrifugation. Repeat the wash Solution (prepared as described below). and centrifugation.

5. Resuspend the cells in 0.5 mL of PBS. DNA Labeling Solution 1 Assay 5 Assays 10 Assays TdT Reaction Buffer 10 µL 50 µL 100 µL 6. Add cells to 5 mL of ice-cold 70% (v/v) ethanol. Let cells stand for a minimum of 30 TdT Enzyme 0.75 µL 3.75 µL 7.5 µL minutes in ice or the freezer. See note below. Br-dUTP 8 µL 40 µL 80 µL

Distilled H2O 32.25 µL 161.25 µL 322.5 µL 7. Store cells in 70% (v/v) ethanol at -20°C until use. Cells can be stored at -20°C several days before use. Total Volume 51 µL 255 µL 510 µL

Note: In some biological systems storage of the cells at -20°C in 70% (v/v) ethanol for at least 12-18 hours prior to staining for apoptosis detection yields the best results. The appropriate volume of Staining Solution to prepare for a variable number of assays is based upon multiples of the component volumes combined for 1 assay. Mix only enough DNA Labeling Solution to complete the number of assays prepared per session. The DNA Labeling Solution is active for approximately 24 hours.

5. Incubate the cells in the DNA Labeling Solution for 60 minutes at 37°C in a temperature controlled bath. Shake cells every 15 minutes to resuspend.

NOTE: The DNA Labeling Reaction can also be carried out at 22-24°C overnight for the control cells. For samples other than the control cells provided in the kit, incubation times at 37°C may need to be adjusted to longer or shorter periods depending on the characteristics of the cells supplied by the researcher.

6. At the end of the incubation time, add 1.0 mL of Rinse Buffer to each tube and centrifuge each tube (300 x g) for five minutes. Remove the supernatant by aspiration.

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 7. Repeat the cell rinsing (as in step 6) with 1.0 mL of the Rinse Buffer (red cap), cen- Analyzing the APO-BRDU™ Samples on the Flow trifuge and remove the supernatant by aspiration. Cytometer 8. Resuspend the cell pellets in 0.1 mL of the Antibody Solution (prepared as described below). This assay is run on a flow cytometer equipped with a 488 nm Argon laser as the light source. Propidium Iodide (total cellular DNA) and Fluorescein (Apoptotic Cells) are the Antibody Solution 1 Assay5 Assays 10 Assays two dyes being used. Propidium Iodide (PI) fluoresces at about 623 nm and Fluorescein Fluorescein~PRB-1 (orange cap) 5 µL 25 µL 50 µL at 520 nm when excited at 488 nm. No fluorescence compensation is required. Two Rinse Buffer (red cap) 95 µL 475 µL 950 µL dual parameter and two single parameter displays are created with the flow cytometer Total Volume 100 µL 500 µL 1000 µL data acquisition software. The gating display should be the standard dual parameter DNA doublet discrimination display with the DNA Area signal on the Y-axis and the DNA Width on the X-axis (Becton-Dickinson, see Figure 4) or DNA Peak/Integral signal for 9. Incubate the cells with the Fluorescein~PRB-1 Antibody Solution in the dark for 30 Coulter, see Figure 5 on page 29. From this display, a gate is drawn around the non- minutes at room temperature. Hint: Wrap tubes with aluminum foil. clumped cells and the second gated dual parameter display is generated. The normal convention of this display is to put DNA (Linear Red Fluorescence) on the X-axis and the 10. Add 0.5 mL of the Propidium Iodide/RNase A Solution to the tube containing the 0.1 FITC~PRB-1 (Log Green Fluorescence) on the Y-axis (see bottom display next page). Two mL Antibody Staining Solution. single parameter gated histograms, DNA and FITC~PRB-1, can also be added but are not necessary. By using the dual parameter display method, not only are apoptotic cells Note: If the cell density is low, decrease the amount of PI/RNase A solution to 0.3 mL. resolved, but at which stage of the cell cycle they are in is also determined. The Log Green Fluorescence histograms of the control cells should look like Figure 3 below. 11. Incubate the cells in the dark for 30 minutes at room temperature.

12. Analyze the cells in Propidium Iodide/RNase Solution by flow cytometry. Negative Control Cells Positive Control Cells 13. Analyze the cells within 3 hours of staining.

Apoptotic Cells Relative Cell Number Relative Relative Cell Number Relative

0 1 2 3 4 10 10 10 10 10 100 101 102 103 104

Log Green Fluorescence

Figure 3: Flow Cytometry Data of APO-BRDU™ Negative & Positive Control Cells.

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Technical Tips and Frequently Asked Questions About Flow Cytometer Setup for Flow Cytometer Setup for Becton Dickinson Hardware Coulter Hardware the APO-BRDU™ Assay Display 1 Display 1

1. For those researchers using adherent cell line systems, the cells in the supernatant have a higher probability of being apoptotic than do the adherent cells. Save cells in Gate the supernatant for assay prior to trypsinization of the adherent cell layer. DNA Peak

DNA Area 2. Cell fixation using a DNA cross-linking chemical fixative is an important step in ana- Gate lyzing apoptosis. Unfixed cells may lose smaller fragments of DNA that are not chemically fixed in place inside the cell during washing steps. The researcher may have to explore alternative fixation and permeabilization methods to fully exploit DNA Width DNA Int. Gated From Display 1 Gated From Display 1 their systems.

3. A cytospin or centrifugal cytology slide can be prepared from APO-BRDU™ samples Positive Apoptotic Positive Apoptotic Cells in the following manner. After completion of the Fluorescein~PRB-1 antibody stain- Cells ing , but prior to the Propidium Iodide/RNAse A treatment, put a drop of the stained cells on a slide, spin it and observe the sample under a fluorescence microscope. Non-Apoptotic Non-Apoptotic Cells Cells FITC ~PBR-1FITC FITC ~PBR-1FITC 4. Surface marker staining of cellular antigens can be accomplished by first incubating the cells with the fluorescent-labeled CD antibody, followed by fixing and perme- abilizing the cells prior to testing in the APO-BRDU™ Assay. DNA Area DNA Int.

TM TM 5. To minimize cell loss during the assay, restrict the assay to the use of a single 12 x Typical FACScan Gain Settings Typical XL Gain Settings Parameter Amplifier Gain Detector Gain Parameter Amplifier Gain Detector Gain 75 mm test tube. If polystyrene plastic test tubes are used, an electrostatic charge FL 1 LogTM 380 Volts FL 1 LogTM 589 Volts can build up on the sides of the tube. Cells will adhere to the side of the tube and FLFigure 3 4: APO-BRDU 1.46Positive Control 414 Volts Cells FigureFL 3 5: APO-BRDU 2.00Positive Control 698 VoltsCells FL 3 Width .87 AUX(FL3 Peak) 1.00 250 Volts the sequential use of multiple tubes can result in significant cell loss during the assay. FL 3 Area 3.25 Discriminator-AUX (FL3 Peak) Threshold- FL 3, 40 6. Occasionally a mirror image population of cells at lower intensity is observed in the flow cytometry dual parameter display. This population arises because during the 50 µL DNA Labeling Reaction, some cells have become stuck to the side of the test tube and are not fully exposed to the reaction solution. This phenomenon can be over- come by washing all the cells from the side of the tube and making sure all cells are properly suspended at the beginning of the labeling reaction.

7. If a low intensity of fluorescein staining is observed, try increasing the incubation time during the 50 µL DNA Labeling Reaction. Some researchers have found labeling times of up to four hours at 37° C may be required for certain cell systems.

8. If the DNA cell cycle information is not required, it is not necessary to add the PI/RNase A solution to each tube.

Product Catalog # Size APO-BRDU™ Assay (TUNEL) KHO1001 60 Tests

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Appendix: Other Apoptosis Products TNF Superfamily Product Description Part Number Size Adaptor & Regulatory Anti-CD40L (CD154), (mouse, Product Description Part Number Size clone MR1) AMT5411x 0.5 mg Anti FADD (human, clone 1F7) AHR5032 100 µg Anti-CD40L (CD154), (mouse, Anti-I-FLICE (human, polyclonal) AHZ0102 100 µg clone MR1, azide-free) AMT5412x 0.5 mg Anti-TRADD (human, clone 3E11) AHR5042 100 µg Anti-CD40L (CD154), (mouse, clone MR1, biotin conj.) AMT5419 0.25 mg Bcl-2 Family Product Description Part Number Size Anti-CD40L (CD154), (mouse, clone MR1, FITC conj.) AMT5418 0.25 mg Anti-Bag (human, mouse, clone 3.9F1E11) AHO0392 100 µg Anti-CD40L (CD154), (mouse, Anti-Bak (human, mouse, polyclonal) AHO0252 100 µg clone MR1, PE/Cy5 conj.) AMT5410 0.1 mg Anti-Bax (human, mouse, polyclonal) AHO0262 100 µg Anti-CD40L (CD154), (mouse, Anti-Bax (human, mouse, rat, clone 6A7) AHO0232 100 µg clone MR1, R-PE conj.) AMT5417 0.1 mg Anti-Bcl-2 (human, clone 124) AHO0042 200 µg Anti-Fas Ligand (human, clone ALF1.2) AHU0212 100 µg Anti-Bcl-2 (human, clone 100/D5) AHO0272 100 µg Anti-Fas Ligand (human, clone ALF1.2, Anti-Bcl-2 (mouse, clone 10C4) AMO0282 100 µg biotin conj.) AHU0219 100 Tests Anti-Bcl-xl (human, rat, mouse, clone 2H12) AHO0222 100 µg Anti-NGRF (human, clone NGRF5) AHU0302 100 µg Anti-Bcl-xl/xs (human, chicken, polyclonal) AHO0242 100 µg Anti-TNF-α (human, clone B-C7, Anti-Mcl-1 (human, clone RC13) AHO0102 100 µg azide free) AHC3012 0.5 mg Anti-TNF-α (human,clone B-C7, Cytochrome c azide free) AHC3013 1.0 mg Product Description Part Number Size Anti-TNF-α (human, clone 68B6A3) AHC3612 0.5 mg Anti-cytochrome c (human, rat, Anti-TNF-α (human, clone 2B3A6A2) AHC3912 0.5 mg mouse, clone 7H8.2C12) ANN0012 100 µg Anti-TNF-α (human, clone 2B3A6A2, Anti-cytochrome c (human, rat, FITC conj.) AHC3918 0.1 mg mouse, clone 6H2.B4) ANN0022 100 µg Anti-TNF-α (human, clone 2B3A6A2, R-PE conj.) AHC3917 0.1 mg DNA Fragmentation and Repair Anti-TNF-α (human, polyclonal) AHC3812 1.0 mg Product Description Part Number Size Anti-TNF-β (human, clone LTX-21, Anti-DFF45 (human, polyclonal) AHF0252 100 µg azide free) AHC3024 0.2 mg Anti-DNA-PK (human, clone 42-PSC) AHF0282 100 µg Anti-TNF-α (mouse, clone MP6-XT3) AMC3814 0.5 mg Anti-Ku80 (human, monkey, clone J15) AHO0092 100 µg Anti-TNF-α (mouse, clone MP6-XT3, Anti-PARP (human, mouse, rat, polyclonal) AHF0262 100 µg azide free) AMC3812 0.5 mg Anti-TNF-α (mouse, clone MP6-XT22, Other Apoptosis Regulators biotin conj.) AMC3919 0.1 mg α Product Description Part Number Size Anti-TNF- (mouse, clone MP6-XT22, Anti-D4-GDI (human, bovine, polyclonal) AHF0272 100 µg FITC conj.) AMC3918 0.1 mg α Anti-keratin-18 (human, clone DC10) AHE0012 100 µg Anti-TNF- (mouse, clone MP6-XT22, R-PE conj.) AMC3917 0.1 mg Anti-p53 (human, monkey, chicken, α clone DO-7) AHO0142 100 µg Anti-TNF- (mouse, polyclonal, azide-free) AMC3012 0.5 mg Anti-p53 (human, bovine, monkey, α clone DO-1) AHO0152 100 µg Anti-TNF- (rat, polyclonal, azide free) ARC3012 0.5 mg Anti-perforin (human, clone dG9) AHF0292 100 µg α Anti-Rb (human, clone 1F8) AHO0172 100 µg Flexia™ TNF- matched antibody pair (human) CHC1751 40 plates

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Product Description Part Number Size Product Description Part Number Size Cytosets™ TNF-α matched Anti- TNFR II (human, clone 911B3H10) AHR3929 0.1 mg antibody pair (human) CHC1754 40 plates Cytosets™ TNFR I Matched Cytosets™ TNF-α matched antibody pair (human) CHC1764 40 plates antibody pair (mouse) CMC3014 40 plates Cytosets™ TNFR II Matched EASIA TNF-α ELISA kit (human) KAC1751 1 plate antibody pair (human) CHC1774 40 plates Cytoscreen™ TNF-α ELISA kit (human) KHC3012 2 plates EASIA™ TNFR I (1 plate), (human) KAC1761 96 Tests Cytoscreen™ TNF-α UltraSensitive TNFR II (1 plate), (human) KAC1771 96 Tests ELISA kit (human) KHC3013 2 plates Cytoscreen™ sFas, 2 plates (human) KHS9502 96 Tests Cytoscreen™ TNF-α ELISA kit (mouse) KMC3012 2 plates Cytoscreen™ TNF-α ELISA kit (rat) KRC3012 2 plates Caspase-1 Peptides Cytoscreen™ TNF-α UltraSensitive Product Description Part Number Size ELISA kit (rat) KRC3013 2 plates Caspase-1 substrate 77-176 0.5 mg Cytoscreen™ TNF-α ELISA kit (primate) KPC3012 2 plates 77-177 1 mg Cytoscreen™ TNF-α ELISA kit (swine) KSC3012 2 plates Ac-YVAD-pNA (substrate color) 77-328 5 mg Cytotrap™ TNF-α Stimulation Assay (human) DHC6752 2 plates 77-330 25 mg Primescreen™ TNF-α primer pair (human) GHC3012 2.5 nmole Ac-YVAD-AMC (substrate fluor) 77-332 5 mg Primescreen™ TNF-α primer pair (mouse) GMC3011 2.5 nmole 77-333 25 mg CytoXpress™ Quantitative mRNA Z-YVAD-pNA (cell perm substrate color) 77-873 5 mg TNF-α kit (human) QHC3012 24 quant. Ac-YVAD-AFC (substrate fluor) 77-876 5 mg CytoXpress™ Quantitative mRNA 77-877 25 mg TNF-α kit (mouse) QMC3012 24 quant. Ac-YVAD-CHO (inhibitor) 77-925 5 mg CytoXpress™ Quantitative mRNA 77-926 25 mg TNF-α kit (rat) QRC3012 24 quant. Z-YVAD-AFC (cell perm substrate fluor) 77-928 5 mg Recombinant NGF-beta (human) PHG0124 10 µg 77-929 25 mg Recombinant TNF-α (human) PHC3015 10 µg YVAD-CHO (cell perm inhibitor) 77-960 1 mg Recombinant TNF-β (human) PHC3024 10 µg 77-961 5 mg Recombinant TNF-α (mouse) PMC3014 10 µg Ac-WEHD-pNA (substrate color) 77-974 5 mg Recombinant TNF-α (rat) PRC3014 10 µg Ac-WEHD-AMC (substrate fluor) 77-978 25 mg

TNFR Superfamily Product Description Part Number Size Anti-FAS (APO-1), (human, clone SM1/1) AHS9502 100 µg Anti-FAS (APO-1), (human, clone B-G27) AHS9501 100 µg Anti FAS (APO-1), (human, clone DX-2) AHS9542 100 µg Anti-FAS (APO-1), (human, clone DX-2/DX-3, FITC conj.) AHS9538 100 Tests Anti-FAS (APO-1), (human, clone 2R2) AHS9552 100 µg Anti-CD40 (human, clone EA-5) AHS4002 100 µg Anti-CD40 (human, clone EA-5, biotin conj.) AHS4009 100 Tests Anti-DR-3 (human, mouse, rat) AHR5002 100 µg Anti-DR-4 (TRAIL-R1), (human) AHR5012 100 µg Anti-DR-5 (TRAIL-R2), (human) AHR5022 100 µg Anti-TNFR I (human, clone 943BC15C12) AHR3819 0.1 mg Anti-TNFR I (human, clone 943C51G1) AHR3912 0.5 mg Anti-TNFR II (human, clone 897C2G9) AHR3022 0.5 mg

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Caspase-2 Peptides Caspase-6 Peptides Product Description Part Number Size Product Description Part Number Size Z-VDVAD-AFC (cell perm substrate fluor) 77-843 5 mg Ac-VEID-pNA (substrate color) 77-861 5 mg Ac-DEHD-AMC (substrate fluor) 78-103 5 mg 77-862 25 mg 78-104 25 mg Z-VEID-AFC (cell perm substrate fluor) 77-864 5 mg Ac-VDVAD-pNA (substrate color) 78-133 5 mg 77-865 25 mg 78-134 25 mg Ac-VEID-AFC (substrate fluor) 77-867 5 mg Ac-VEHD-AMC (substrate fluor) 78-106 5 mg Caspase-3 Peptides 78-107 25 mg6 Product Description Part Number Size VEID-CHO (cell perm inhibitor) 78-119 5 mg Z-DEVD-pNA (cell perm substrate color) 77-870 5 mg Ac-DEVD-OH 77-897 5 mg Caspase-9 Peptides 77-898 25 mg Product Description Part Number. Size Ac-DEVD-pNA (substrate color) 77-900 5 mg Ac-VEHD-AMC (substrate fluor) 78-106 5 mg Ac-DEVD-AMC (substrate fluor) 77-910 5 mg 78-107 25 mg Ac-DEVD-CHO (inhibitor) 77-920 5 mg Ac-LEHD-AMC (substrate fluor) 78-109 5 mg 77-921 25 mg Ac-LEHD-pNA (substrate color) 78-136 5 mg Z-DEVD-AFC (cell perm substrate fluor) 77-931 5 mg 78-137 25 mg Ac-DEVD-AFC (substrate fluor) 77-934 5 mg Granzyme B Peptides DEVD-CHO (cell perm inhibitor) 77-950 1 mg Product Description Part Number Size 77-951 5 mg Ac-IETD-CHO (inhibitor) 77-849 5 mg MCA-DEVDAR[K-DNP]-amide 77-966 0.5 mg 77-850 25 mg (FRET peptide) 77-967 1 mg Z-IETD-pNA (cell perm substrate color) 77-852 5 mg Ac-VAD-CHO (pan caspase inhibitor) 77-972 1 mg 77-853 25 mg 77-973 5 mg Ac-IETD-pNA (substrate color) 77-855 5 mg Ac-IETD-AFC (substrate fluor) 77-980 5 mg Caspase-4 Peptides Z-IETD-AFC (cell perm inhibitor fluor) 77-983 5 mg Product Description Part Number Size 77-984 25 mg Ac-LEVD-CHO (inhibitor) 77-846 5 mg Ac-IETD-AMC (substrate fluor) 78-100 5 mg 77-847 25 mg 78-101 25 mg MCA-LEVDGW[K-DNP]-amide 77-882 0.5 mg IETD-CHO (cell perm inhibitor) 78-113 5 mg (FRET peptide) LEVD-CHO (cell perm inhibitor) 78-115 1 mg 78-116 5 mg

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Apoptosis-Multiplex mRNA Kits Human Kits References Product Description Part Number Size Apoptosis Set 1: Caspase 1, Bcl-2, p53, Annexin-V: c-myc, GAPDH QHM0011 50 tests 1. Boersma, A.W., et al. (1996) Quantification of apoptotic cells with fluorescein isothiocyanate- Apoptosis Set 2: Bax, Bcl-2, Bcl-xS, labeled Annexin-V in Chinese hamster ovary cell cultures treated with cisplatin. Cytometry Bcl-XL, LICE (Caspase-3), GAPDH QHM0021 50 tests 24:123-130. Apoptosis Set 3: Fas, Fas L, TRAIL, 2. Homburg, C.H., et al. (1995) Human neutrophils lose their surface Fc gamma RIII and acquire Caspase-8, GAPDH QHM0031 50 tests Annexin-V binding sites during apoptosis in vitro. Blood 85:532-540. 3. Martin, S.J., et al. (1995) Early redistribution of plasma membrane phosphatidylserine is a gen- Apoptosis Set 4: Fas, Fas L, FLICE, FADD, eral feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of TRADD, GAPDH QHM0041 50 tests Bcl-2 and Abl. J. Exp. Med. 182:1545-1556. Apoptosis Set 5: Apaf-1, Caspase-3, 4. Vermes, I., et al. (1995) A novel assay for apoptosis: flow cytometric detection of phos- Caspase-5, Caspase-8, Caspase-9, GAPDH QHM0051 50 tests phatidylserine expression on early apoptotic cells using fluorescein labeled Annexin-V. Apoptosis Set 6: Caspase-2, Caspase-4, J. Immunol. Methods 184:39-51. Caspase-6, Caspase-7, Caspase-10, GAPDH QHM0174 50 tests Caspase: Mouse Kits 1. Casciola-Rosen, L.A., et al. (1996) Apopain/CPP32 cleaves proteins that are essential for cel- Product Description Part Number Size lular repair: a fundamental principle of apoptotic death. J. Exp. Med. 183(5):1957-1964. Apoptosis Set 1: Caspase-1, Bcl-2, p53, 2. Lazebnik, Y., et al. (1994) Cleavage of poly (ADP-ribose) polymerase by a proteinase with c-myc, GAPDH QMM0011 50 tests properties like ICE. Nature 371(6495):346-347. 3. Jaeschke, H., et al. (1998) Activation of Caspase-3 (CPP32)-like proteases is essential for TNF- Apoptosis Set 2: Bax, Bcl-2, Bcl-XS, α-induced hepatic parenchymal cell apoptosis and neutrophil-mediated necrosis in a murine Bcl-XL, LICE (Caspase-3), GAPDH QMM0021 50 tests endotoxin shock model. J. Immunol. 160(7):3480-3486. Rat Kits 4. Anel, A., et al. (1997) Inhibition of CPP32-like proteases prevents granzyme B- and Fas-, but not granzyme A-based cytotoxicity exerted by CTL clones. J. Immunol. 158(5):1999-2006. Product Description Part Number Size 5. Talanian, R.V., et al. (1997) Substrate specificities of caspase family proteases. J. Biol. Chem. Apoptosis Set 1: Caspase-1, Bcl-2, p53, 272(15):9677-9682. c-myc, GAPDH QRM0011 50 tests 6. Stennicke, H.R., et al. (1997) Biochemical characteristics of Caspases-3, -6, -7 and -8. J. Biol. Apoptosis Set 2: Bax, Bcl-2, Bcl-XS, Chem. 272(41):25719-25723. Bcl-XL, LICE (Caspase-3), GAPDH QRM0021 50 tests DNA Ladder: 1. Wyllie, A.H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 284(5756):555-6. 2. Park, D.J., et al. (1998) Detergent and enzyme treatment of apoptotic cells for the observation of DNA fragmentation. Biotechniques 24(4):558-60. TUNEL 1. Li, X. and Darzynkiewicz, Z. (1995) Labeling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Prolif. 28:572-579. 2. Li, X., Traganos, F., Melamed, M.R. and Darzynkiewicz, Z. (1995) Single step procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleotides. Detection of apoptosis and BrdUrd incorporation. Cytometry 20:172-180. 3. Goldman, A.S., Baker, M.K. and Piddington, R. (1983) Inhibition of programmed cell death in mouse embryonic palate in vitro by cortisol and phenytoin: receptor involvement and require- ments of protein synthesis. Proc. Soc. Exp. Biol. Med. 174:239-243. 4. Lockshin, R.A. and Zakeri, Z. Programmed cell death and apoptosis. In: L. D. Tomei and F. D. Cope (eds.), Current Communications in Cell and Molecular Biology, Vol. 3., pp. 47-60. Cold Springs Harbor, NY: Cold Springs Laboratory, 1991. 5. Buttyan, R., Zakeri, R., Lockshin, R.A. and Wolgemuth, D. (1988) Cascade induction of c-fos, c-myc and heat shock 70k transcripts during regression of the rat ventral prostate gland. Mol. Endocrinol. 2:650-658. 6. Weedon, D. and Strutton, G. (1981) Apoptosis as the mechanism of the involution of hair folli- cles in catagen transformation. Acta Derm. Venerol. 61:335-343.

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Apoptosis Glossary (version 1.3) Apoptosis: 7. Strasser, A., Harris, A.W. and Cory, S.(1991) bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Cell 67:889-899. Greek word meaning the dropping of leaves from a tree. 8. Hasbold, J. and Klaus, G.G.B. (1990) Anti-immunoglobulin antibodies induce apoptosis in Describes the common morphological changes that characterize the immature B cell lymphomas. Eur. J. Immunol. 20:1685-1690. process of cellular self-destruction. 9. Appleby, D.W. and Modak, S.P. (1977) DNA degradation terminally differentiating lens fiber cells from chicken embryos. Proc. Natl. Acad. Sci. USA 74:5579-5583. 10. Benedetti, A., Jezequel, A.M. and Orland, I. (1988) Preferential distribution of apoptotic bod- Glossary ies in acinar zone 3 of normal and rat liver. J. Hepatol. 7:319-324. 4-1BB Also known as CD137. 4-1BB is a member of the TNF receptor family and induces 11. Thompson, C.B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science apoptosis in T cells (Proc Natl Acad Sci USA 1989, 86:1963-1967; J Immunol 1998, 267:1456-1462. 160:2488). 12. Arends, M.J., Morris, R.G. and Wyllie, A.H. (1990) Apoptosis: the role of endonuclease. Am. J. Pathol. 136:593-608. A 13. Darzynkiewicz, Z., Li, X. and Gong, J. Assays of cell viability. Discrimination of cells dying in A1 A Bcl-2 family member that is also known as BFL-1 protein. A1 is expressed in hemo- apoptosis in Methods in Cell Biology: Flow Cytometry, 2nd edition., Darzynkiewicz, Z., poietic tissues, prolongs cell survival, and permits myeloid differentiation (J Immunol Crissman, H.A. and Robinson, J.R., eds., Academic Press, 1994. 1993, 151:1979-1988; Blood 1996, 87:983-992). 14. Steller, H. (1995) Mechanisms and gene of cellular suicide. Science 267:1445-1449. A20 A20 is a cytokine-induced primary response gene that encodes a protein that inhibits 15. Nagata, S. and Golstein, P. (1995) The Fas death factor. Science 267:1449-1456. apoptosis (J Biol Chem 1990, 265:14705-14708; J Immunol 1995, 154:1699-1706). 16. Eschenfeldt, W.H., Puskas, R.S. and Berger, S.L. Homopolymeric tailing in Methods in AIF Apoptosis inducing factor (FEBS Lett 1998, 427:198-202). Enzymology, 152:337-342, Berger, S.L. and Kimmel, A.R., eds., Academic Press, 1987. AIP1 Apoptosis inducing protein; a protein associated with the apoptosis-linked gene 17. Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M., Lassota, P. and ALG2 (J Biol Chem 1999, 274:1533-1540). Traganos, F. (1992) Features of apoptotic cells measured by flow cytometry. Cytometry Akt Also known as protein kinase B or PKB. Akt is a serine/threonine kinase, that prevents 13:795-808. apoptosis of neurons (Science 1997, 275:628- 630). ALG-2 Apoptosis-linked gene-2. A pro-apoptosis gene that codes for a Ca++ binding protein required for T cell receptor and Fas induced cell death. ALG-2 is also involved in neuronal survival (Science 1996, 271:521-525). ALG-3 Apoptosis-linked gene-3. An Alzheimer’s Disease gene that protects PC12 cells from glutamate induced apoptosis (J Biol Chem 1996, 271:31025-31028). Annexin-V A phosphatidylserine binding protein that may be used for detecting apoptotic cells. Apaf-1 Apoptosis protease activating factor-1. A human protein homologous to C. elegans CED-4 that participates with Caspase-9 ( Apaf-3) in the cytochrome c-dependent activation of Caspase-3, leading to apoptosis (Cell 1997, 90:405-413). Apaf-2 Apoptosis protease activating factor-2 (Also known as Cytochrome c). Apaf-3 Apoptosis protease activating factor-3 (Also known as Caspase-9) (J Biol Chem 1996, 271:16720-16724). APO-1 Also known as Fas and CD95, a member of the TNF receptor family. Cross-linking APO-1 induces apoptosis (J Biol Chem 1992, 267:10709-10715). APO-1L APO-1 ligand, also known as fas ligand. A member of the TNF family that induces apoptosis (Cell 1993, 75:1169-1178). APO-2 Receptor for TRAIL/APO-2L. A member of the TNF receptor family (Curr Biol 1998, 8:113-116). APO-2L APO-2 ligand that is also known as TRAIL. APO-2L is a member of the TNF family that induces apoptosis (J Biol Chem 1996, 271:12687-12690). APO-3 A death domain containing receptor. Also known as DR3, WSL-1, TRAMP or LARD (Curr Biol 1996, 6:1669-1676). APO-3L The ligand for the death-domain-containing receptor Apo-3. The Apo-3 ligand is a 249 amino-acid, type II transmembrane protein that induces apoptosis and NF-κB activation (Curr Biol 1998 8:525-528 ). Apopain Another name for Caspase-3. (Nat Struct Biol 1996, 3171:619-625).

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Apoptosis Greek word meaning the dropping of leaves from a tree. Describes the common mor- Bik Bcl-2 interacting killer. Bik is a BH-3 domain containing protein that is also known phological changes that characterize the process of cellular self-destruction (Br J as BP4, Bip1 and NBK. Bik is pro-apoptosis (Oncogene 1995, 11:1921-1928). Cancer, 1972, 26:239-245). Bim A member of the Bcl-2 family that promotes apoptosis (EMBO J 1998, 17:384-395). Apoptosome The complex whose assembly is central to the activation of apoptosis and that comprises Bip-1 Another name for Bik. the vertebrate homologue of the nematode. Ced-4 protein, Apaf-1, holo-cytochrome c BIR Motif A baculovirus IAP repeat motif that is involved in inhibiting apoptosis. (released from mitochondria), deoxy-adenosinetriphosphate, and pro-Caspase-9 Blk Bik-like killer protein. Blk is a BH3-containing mouse protein that interacts with Bcl-2 (Science 1998, 281:1317-1322). and Bcl-XL. Blk is a potent death agonist (J Biol Chem 1998, 273:7783-7786). ARC Apoptosis repressor with caspase recruitment domain. ARC is expressed primarily in BOD Bcl-2-related ovarian death gene, an ovarian BH3 domain-containing pro-apoptotic skeletal muscle and cardiac tissue and inhibits apoptosis (Proc Natl Acad Sci USA Bcl-2 protein. (Mol Endocrinol 1998,12:1432-40). 1998, 95:5156-5160). Bok Bcl-2 related ovarian killer. Bok is a pro-apoptotic Bcl-2 protein with restricted ASK1 Apoptosis signal-regulating kinase-1, also known as MEKK5 or MAPKKK5, induces expression in reproductive tissues that heterodimerizes with selective anti-apoptotic apoptosis (Biochem Biophys Res Commun. 1997, 239:905-910). Bcl-2 family members (Proc Natl Acad Sci USA 1997, 94:12401-12406). Boo Inhibits apoptosis (EMBO J 1999, 18:167-178). B BP4 Another name for Bik. Bad Bcl-xL/Bcl-2 associated death promoter. Bad is a heterodimeric partner for Bcl-xL Btf Bcl-2-associated transcription factor. It interacts with Bcl-2 and Bcl-xL and plays a and Bcl-2 that displaces Bax and promotes cell death (Cell 1995, 80:285-291). role in pro-apoptosis. (Mol Cell Biol 1999, 19:4390-4404). Bag Bcl-2 associated athanogene-1. Bag is a Bcl-2-binding protein which provides pro- tection from apoptotic cell death (Cell 1995, 80:279-284). C Bak Bcl-2 antagonist/killer. Bak is pro-apoptosis (Genomics 1997, 44:195-200). cIAP1 Cellular inhibitor of apoptosis protein 1. One of the molecules that is recruited to the BAR Apoptosis regulator. Bar can bridge Caspase-8 and Bcl2 (PNAS 2000, 97(6):2597- cytosolic domain of TNF-RII via its association with TRAF1 and TRAF2 (Proc Natl 2602). Acad Sci USA 1996, 93:13973-13978). Bax Bcl-2 associated x protein. Bax is a member of the Bcl-2 family and is pro-apoptosis cIAP2 Cellular inhibitor of apoptosis protein 2. One of the molecules that is recruited to the (Cell 1993, 74:609-619). cytosolic domain of TNF-RII via its association with TRAF1 and TRAF2 (Proc Natl Bax α One of the Bax isoforms that promotes apoptosis. The protein is 21 kDa. Acad Sci USA 1996, 93:13973-13978). Bax β One of the Bax isoforms that promotes apoptosis. The protein is 24 kDa. CADA caspase-activated DNase in mouse. CAD is homologous to human CPAN or DFF40 Bax γ One of the Bax isoforms. (Nature 1998, 391:43-50). Bax ω A splice variant of Bax (J Biol Chem 1998, 273:11930-11936). Calpain A non-ICE cysteine proteinase, associated with apoptosis (J Cell Physiol 1994, Bcl-2 B-cell lymphoma 2. Belongs to the Bcl-2 family of proteins and is known to inhibit 159:229-237). apoptosis. CAML Calcium-modulator and cyclophilin ligand. CAML is a co-inducer of NF-AT activation Bcl-10 A CARD-containing protein with homology to herpesvirus-2 protein E10, identified and binds to TACI, a member of TNF receptor superfamily (Nature 1994, 371:355-358; in MALT B cell lymphomas. It is also known as mE10, c-E10, CARMEN, CLAP and Science 1997, 278:138-141). CIPER. It activates NF-κB and is pro-apoptotic (Cell 1999, 96:35-45 and Nat Genet CAP-1 CD40-associated protein-1. CAP-1 specifically binds to the cytosolic domain of 1999, 22: 63-68). CD40 and contains a C-terminal domain that shares strong amino acid sequence Bcl-b A novel Bcl-2 family member that regulates Bax and Bak (J Biol Chem 2000, homology to TRAF1 and TRAF2 (FEBS Lett 1995, 358:113-118). 276(16):12481-12484). CARD Caspase activation and recruitment domain. Bcl-g A novel member of the Bcl-2 family. Bcl-g is pro-apoptotic (J Biol Chem 2000, CARD4 A CED4/Apaf-1-like molecule that can regulate both apoptosis and NF-κB activation 276(4):2780). pathways. (J Biol Chem 1999, 274:12955-12958). Bcl-w A member of the Bcl-2 family that promotes cell survival (Oncogene 1996, 13:665-675). CARDIAK CARD-containing ICE associated kinase, also known as RICK and RIP2, involved in Bcl-x A Bcl-x isoform that inhibits apoptosis (Biochem Biophys Res Commun 1998, NFκ B/JNK signaling and in the generation of the proinflammatory cytokine IL-1 beta 248:147-152). through activation of Caspase-1 (Curr Biol 1998, 8:885-888). κ Bcl-xL Long form of Bcl-x. A Bcl-2-related gene that functions as a dominant regulator of CARMEN CARD containing molecule enhancing NF- B, also known as Bcl-10, c-E10, CIPER, κ apoptotic cell death. Bcl-xL inhibits cell death (Cell 1993, 74:597-608). CLAP and mE10. It activates NF- B and is pro-apoptotic. (J Biol Chem 1999, 274:9962-9968) Bcl-xS Short form of Bcl-x (BH1 and BH2 are spliced out). Bcl-xS is pro-apoptosis. Beclin A Bcl-2-interacting protein (J Virol 1998, 72:8586-96). CART1 Cysteine rich domain associated to ring and traf protein. CART1 is also known as Bfl-1 A Bcl-2 homolog that has been isolated from a human fetal liver. Bfl-1, also known TRAF4 and is a novel member of the tumor necrosis factor receptor-associated pro- as A1, is anti-apoptosis (Oncogene 1995, 11:1693- 1698). tein family (J Biol Chem 1995, 270:25715-25721). α BH1 Bcl-2 homolog domain-1 (Adv Exp Med Biol 1996, 406:99-112). CAS Cellular apoptosis susceptibility protein. Plays a role in toxin or TNF- induced BH2 Bcl-2 homolog domain-2 (Adv Exp Med Biol 1996, 406:99-112). apoptosis (Proc Natl Acad Sci USA 1995, 92:10427-10431). BH3 Bcl-2 homolog domain-3 (Adv Exp Med Biol 1996, 406:99-112). Caspase Cysteinyl aspartic acid-protease (Human ICE/CED3 nomenclature (Cell 1996, 87:171). BH4 Bcl-2 homolog domain-4 (Adv Exp Med Biol 1996, 406:99-112). Caspase-1 Cysteinyl aspartic acid-protease-1. Caspase-1 is also known as ICE and is a CED-3 BI-1 Bax inhibitor-1 that suppresses apoptosis induced by Bax (Mol Cell 1998, 1:337-346). homologue (Nature 1992, 356:768-774). Bid BH-3 Interacting Domain Death agonist that induces ICE-like proteases and apoptosis Caspase-2 Cysteinyl aspartic acid-protease-2. Caspase-2 is also known as Ich-1 and Nedd2 (Cell (Genes Dev 1996, 10:2859-2869). 1994, 78:739-750).

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 Caspase-3 Cysteinyl aspartic acid-protease-3. Caspase-3 is also known as CPP32, Yama, CPP32 Also known as Caspase-3 (J Biol Chem 1994, 269:30761-30764). Apopain, SCA-1, and LICE (J Biol Chem 1994, 269:30761-30764). CRADD Caspase and RIP adaptor with death domain. CRADD is involved in apoptosis sig- Caspase-4 Cysteinyl aspartic acid-protease-4. Caspase-4 is also known as ICEreI-II, TX and ICH-2 naling in response to TNF-RI stimulation (Cancer Res 1997, 57:615-619). (EMBO J 1995, 14:1914-1922). CrmA Cytokine response modifier A is a viral product that inhibits apoptosis. Caspase-5 Cysteinyl aspartic acid-protease-5. Caspase-5 is also known as ICErel-III and TY (J Cytochrome c An electron carrier in the cell respiratory chain that plays a critical role in apoptosis Biol Chem 1995, 270:15870-15876). (Cell 1996, 86:147-157).) Caspase-6 Cysteinyl aspartic acid-protease-6. Caspase-6 is also known as Mch-2 (Cancer Res 1995, 55:2737-2742). D Caspase-7 Cysteinyl aspartic acid-protease-7. Caspase-7 is also known as Mch-3, ICE-LAP-3 D4-GDI GDP-Dissociation Inhibitor for Ras-related Rho family GTPase. D4-GDI is a substrate and CMH-1 (Cancer Res 1995, 55:6045-6052). for Caspase-3 (Eur J Immunol 1998, 28:296-304). Caspase-8 Cysteinyl aspartic acid-protease-8. Caspase-8 is also known as FLICE, Mach-1 and DAP kinase Death associated protein kinase. DAP kinase is a mediator of apoptosis induced by Mch5 (Cell 1996, 85:817-827). IFN-γ (Mol Cell Biol 1998, 8:1642-1651). Caspase-9 Cysteinyl aspartic acid-protease-9. Caspase-9 is also known as ICE-LAP6, Mch6 and DAP-1 Death associated protein-1. DAP-1 is a 15 kDa protein and a mediator of gamma Apaf-3 (J Biol Chem 1996, 271:16720-16724). interferon-induced cell death (Genes Dev 1995, 9:15-30). Caspase-10 Cysteinyl aspartic acid-protease-10. Caspase-10 is also known as FLICE-2 and Mch4 DAP-3 Death associated protein-3. DAP-3 is a mediator of gamma interferon induced cell (J Biol Chem 1997, 272:6578-6583). death (J Biol Chem 1995, 270:27932-27936). Caspase-11 Cysteinyl aspartic acid-protease-11 (FEBS Lett 1997, 403:61-69). DAP-5 Death associated protein-5. DAP-5 is a mediator of gamma interferon induced cell Caspase-12 Cysteinyl aspartic acid-protease-12 (FEBS Lett 1997, 403:61-69). death (Mol Cell Biol 1997, 17:1615-1625). Caspase-13 Cysteinyl aspartic acid-protease-13. Caspase-13 is a FLICE activatable caspase, and Daxx Daxx is a Fas-binding protein. Over-expression of Daxx enhances Fas-mediated is also known as ERICE (J Biol Chem 1998, 273:15702-15707). apoptosis (Cell 1997, 89:1067-1076). Caspase-14 Cysteinyl aspartic acid-protease-14, also know as MICE. Overexpression of MICE DcR-1 Decoy receptor-1. DcR-1 is a receptor for TRAIL that inhibits TRAIL signaling. DcR-1 induces apoptosis (J Biol Chem 1998, 273:29648-29653 and Cancer Res 1998, is also known as TRID, LIT and TRAIL-R3 (Science 1997, 277:815-818). 58:5201-5205). DcR-2 Decoy receptor 2. DcR-2 is the receptor for Trail that contains a truncated death Casper A FADD- and caspase-related inducer of apoptosis known as FLIP (Immunity 1997, domain and functions as an inhibitory Trail receptor. DcR-2 is also known as TRAIL-R4 6:751-763). (Curr Biol 1997, 7:1003-1006). CD40 A member of the tumor necrosis factor receptor superfamily that regulates B-lym- DcR-3 Decoy receptor 3. It binds to FasL and inhibits FasL-induced apoptosis, (Nature phocyte proliferation, immunoglobulin class-switching, and apoptosis (FEBS Lett 396:699-703.) 1995, 352:113-118). DD Death domain. CD40L CD40 ligand. CD40L is also known as CD154 and TRAP (Eur J Immunol 1992, Death Effector 22:3191-3194). Filaments Cytoplasmic structures that recruit caspases and trigger apoptosis (J Cell Biol 1998, c-E10 A CARD-containing protein with homology to herpesvirus-2 protein E10, also known 141:1243-1253). as Bcl-10, CARMEN, CIPER, c-E10 and mE10. It activates NF-κB and is pro-apoptotic DED Death Effector Domain. (J Biol Chem 1999, 274:20127-20132). DEDD A death effector domain-containing protein, induces apoptosis (EMBO 1998, CED-1 A cell death gene in the nematode C. elegans (Brain Pathol 1996, 6:411-425). 17:5974-86). CED-3 A cell death gene in the nematode C. elegans. CED-3 promotes apoptosis and is a DEFT Death Effector domain-containing Testicular molecule, highly expressed in testis homologue to Apaf-3/Caspase-9 and Caspase-3 (J Biol Chem 1994, 269:30761-30764). germ cells (Endocrinology 1998, 139:4839-4848). CED-4 A cell death gene in Caenorhabditis elegans. CED-4 is a cell death activator and a DEHD-AMC A synthetic tetrapeptide DEHD (Asp-Glu-His-Asp) conjugated to 7-amino-4-methyl homologue to Apaf-1 (Cell 1997, 90:405-413). coumarin, DEHD-AMC is a fluorogenic substrate for Caspase-2. CED-4L The longer form of CED4 that inhibits apoptosis. DEVD Amino acid residues (Asp-Glu-Val-Asp) of Caspase-3 cleavage site within PARP. CED-4S The short form of CED4 that induces apoptosis (Brain Pathol 1996, 6:411-425). DEVD-AFC A tetrapeptide Asp-Glu-Val-Asp conjugated to 7-amino-4- trifluoromethyl coumarin, CED-9 A cell death gene in nematode C. elegans. CED-9 belongs to the Bcl-2 family and is a fluorometric substrate for caspase enzyme activity assay. also known as cell death protein 9 (Cell 1994, 76:665-676). DEVD-CHO Asp-Glu-Val-Asp-aldehyde. DEVD-CHO is a synthetic Caspase-3 inhibitor. CIDE A novel family of cell death activators with homology to the 45 kDa subunit of the DEVD-FMK Asp-Glu-Val-Asp-O-methyl-fluoromethylketone. DEVD-FMK is a synthetic Caspase-3 DNA fragmentation factor (EMBO J 1998, 17:2526-2533). inhibitor. CIPER CED-3/ICH-1 prodomain homologous, E10-like regulator, also known as Bcl-10, c-E10, DEVD-pNA A synthetic tetrapeptide DEVD (Asp-Glu-Val-Asp) conjugated to p-nitroanilide (pNA), CARMEN, CLAP and mE10. It is a CARD-containing protein and pro-apoptotic (J Biol a colorimetric substrate for caspase enzyme activity assay. Chem 1999, 274:9955-9961). DEVD-AMC Asp-Glu-Val-Asp-7-Amino-4-methyl coumarin. DEVD-AMC is a fluorogenic sub- CLAP CARD-like apoptotic protein. Also know as Bcl-10, CARMEN, CIPER, c-E10 and strate for Caspase-3. mE10. (J Biol Chem 1999, 274:17946-54). DFF DNA Fragmentation Factor. DFF is a heterodimer of a 45 kDa (DFF45) and 40 KDa CLARP A death effector domain-containing protein that interacts with Caspase-8 and regu- (DFF40) protein. lates apoptosis. Also known as FLIP (Proc Natl Acad Sci USA 1997, 94:10717-10722). DFF40 A 40 kDa subunit of DFF. DFF40 is the active component of DFF and triggers DNA CPAN Caspase activated nuclease. CPAN is also known as DFF40, a subunit of DFF, that fragmentation during apoptosis (Proc Natl Acad Sci USA 1998, 95:8461-8466). degrades DNA and induces DNA fragmentation. CPAN is homologous to mouse DFF45 A 45 kDa subunit of DFF. DFF45 is also know as ICAD. During apoptosis, Caspase-3 CAD (Curr Biol 1998, 8:537-540). cleaves DFF45 which then dissociates from DFF40 (Cell 1997, 89:175-184). e-mail: [email protected] Technical/Customer Services 1-800-242-0607 DISC Death inducing signaling complex. G Diva A Bcl-2 homologue, binds to apaf-1 and induces BH3-independent cell death (J Biol GITR Glucocorticoid-induced TNF receptor family related gene. GITR is a member of the Chem. 1998, 273:32479-86). tumor necrosis factor/nerve growth factor receptor family that inhibits T cell recep- DNA-PK DNA-dependent protein kinase. DNA-PK is involved in DNA double-strand-break tor-induced apoptosis (Proc Natl Acad Sci USA 1997, 94:6216-6221). repair. Granzyme A A serine protease located in the granules of cytotoxic T cells and NK cells that is DNA-PKcs DNA-dependent protein kinase catalytic site. DNA-PKcs is a catalytic domain of involved in the induction of target cell apoptosis (J Immunol 1988, 141:3471-3477). DNA-PK. Granzyme B A serine protease located in the granules of cytotoxic T cells and NK cells that is DP5 Neuronal death protein. DP5 is a death promoting gene (J Biol Chem 1997, involved in the induction of target cell apoptosis (J Immunol 1988, 141:3471-3477). 272:18842-18848; Brain Res Mol Brain Res 1998, 54:316-320). DR-3 Death receptor-3. DR-3 is a member of the TNF receptor family that is also known as Apo-3, WSL-1, TRAMP or LARD (Science 1996, 274:990- 992). H DR-4 Death receptor-4. One of the receptors for TRAIL that is also known as TRAIL-R1 Hdaxx Human homologue to murine Daxx. Hdaxx binds to the death domain of Fas (CD95) (Science 1997, 276:111-113). (J Cell Sci 1998, 111:2029-2041). DR-5 Death receptor-5. One of receptors for TRAIL that is also known as TRAIL-R2 HIAP1 Human inhibitor of apoptosis protein 1. HIAP1, also known as c-IAP2, is a member (Immunity 1997, 7:821-830). of the IAP family and is an apoptotic suppressor (Cell 1995, 83:1243-1252). DR-6 Death receptor-6, DR6 interacts with TRADD, which has previously been shown to HIAP2 Human inhibitor of apoptosis protein 2. HIAP2, also known as c-IAP1, is a member associate with TNFR1. It induces apoptosis and activation of both NF-κB and JNK of IAP family and an apoptotic suppressor (Proc Natl Acad Sci USA 1996, 93:13973- (FEBS Lett 1998, 431:351-356). 13978). DRAK1 DAP kinase-related apoptosis-inducing protein kinase 1, a serine/threonine kinase HILP Human IAP-like protein, regulates programmed cell death downstream of Bcl-xL and related to death-associated protein kinase (J Biol Chem 1998, 273:29066-29071). cytochrome c. Also known as XIAP (Mol Cell Biol 1998, 18:608-615). DRAK2 DAP kinase-related apoptosis-inducing protein kinase 2, a serine/threonine kinase HrK A product of harakiri. Hrk is a member of Bcl-2 family and activates apoptosis related to death-associated protein kinase (J Biol Chem 1998, 273:29066-29071). (EMBO J 1997, 16:1686-1694). DRP-1 Pro-apoptotic Ca2+/calmodulin-regulated serine/threonine kinase. DRP-1 is a mem- HVEM Herpesvirus entry mediator, a member of TNF receptor superfamily, mediates herpes- ber of the BAP kinase family (EMBO J 2001, 20(5):1099-1113). virus entry into cells during infection (Cell 1996, 87:427-436). HVEM-L Herpesvirus entry mediator ligand, a ligand for HVEM/TR2, stimulates proliferation E of T cells and inhibits HT29 cell growth (J Biol Chem 1998, 273:27548-56). ERICE A FLICE-activatable caspase. Also known as caspase-13 (J Biol Chem 1998, 273:15702-15707). I Endothelial IAPs Inhibitor of apoptosis proteins. The proteins include NAIP, c-IAP1, c-IAP-2, X-IAP and IL-8 Interleukin-8. IL-8, a neutrophil chemoattractant, can also induce apoptosis survivin (J Virol 1993, 67:2168-2174). (Biochem Biophys Res Comm 1998, 243:407-411). IB Inhibitor of NF-κB. ICAD Inhibitor of caspase-activated DNAse. ICAD is the mouse homologue to human F DFF45 (Nature 1998, 391:43-50). ICE Interleukin-1β converting enzyme. ICE is also known as Caspase-1 (Science 1992, FADD Fas associated death domain. FADD is also known as MORT-1, an apoptotic adaptor 256:97-100). molecule that recruits Caspase-8 or Caspase-10 to the activated Fas or TNFR-I recep- ICE-LAP3 ICE-like apoptotic protease 3. ICE-LAP3 is also known as Caspase-7 (J Biol Chem tors (Cell 1995, 81:505-512). 1996, 271:1621-1625). FAF-1 Fas associated factor-1. FAF-1 potentiates apoptosis. ICE-LAP6 ICE-like apoptotic protease 6. ICE-LAP6 is also known as Caspase-9 (J Biol Chem FAP-1 Fas associated phosphatase-1. FAP-1 is a protein tyrosine phosphatase that associates 1996, 271:16720-16724). with Fas (Science 1995, 268:411-415). ICErel II ICE/CED-3-related protease. ICErel II is also known as Caspase-4 (J Biol Chem 1995, Fas Known as CD95 and APO-1. Fas is a member of the TNF receptor family and pro- 270:15870-15876). motes apoptosis (Cell 1991, 66:233-243). ICErel III ICE/CED-3 related protease. ICErel III is also known as Caspase-5 (J Biol Chem 1995, Fas-DISC Fas-death inducing signaling complex (Nat Med 2001, 7(1):26-27). 270:15870-15876). Fas L Fas ligand. FasL is also known as APO-1 ligand (Cell 1993, 75:1169-1178). Ich-1 ICE and ced-3 homologue-1. Ich-1 is another name for Caspase-2 (Cell 1994, 78:739- FLAME-1 FADD-like anti-apoptotic molecule, inhibitor of Fas/TNF-RI induced apoptosis, also 750). known as FLIP (J Biol Chem 1997, 272:18542-18545) Ich-1L Alternatively spliced Ich-1. Ich-1L codes for 435 amino acids and promotes apoptosis. FLICE Fadd-like ICE. FLICE is also known as Caspase-8 (Cell 1996, 85:817-827). Ich-1s Alternatively spliced Ich-1. Ich-1s codes for 312 amino acids and suppresses apoptosis. FLICE-2 Fadd-like ICE-2. FLICE-2 is also known as Caspase-10 (J Biol Chem 1997, 272:6578-6583). Ich-2 Ice and ced-3 homologue-2. Ich-2 is also known as Caspase-4 (J Biol Chem 1995, FLIP FLICE inhibitory protein. FLIP regulates apoptosis and is also known as I-FLICE, 270:15250-15256). CASH, CLARP, FLAME-1, Casper, and MRIT (Nature 1997, 388:190-195). IETD Ile-Glu-Thr-Asp. The amino acid sequence corresponds to one of the Caspase-8 cleavage sites (amino acids 172-175) of the inactive caspase-3 precursor. IETD-AFC A tetrapeptide, Ile-Glu-Thr-Asp, conjugated to 7-amino-4- trifluoromethyl coumarin, a fluorometric substrate for Caspase-8-like enzyme activity assay.

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 IETD-AMC Ile-Glu-Thr-Asp-7-Amino-4-methyl coumarin. IETD-AMC is a fluorogenic substrate Lymphotoxin Also known as TNF-β (Cell 1993, 72:847-856). for Caspase-8. IETD-CHO Ile-Glu-Thr-Asp-aldehyde. IETD-CHO is a synthetic caspase-8 inhibitor. M IETD-FMK Ile-Glu-Thr-Asp-O-methyl-fluoromethyketone. IETD-FMK is a synthetic Caspase-8- MACH MORT-Associated CED-3 Homologue. MACH is another name for Caspase-8 (Cell like inhibitor. 1996, 85:803-815). IETD-pNA A synthetic tetrapeptide DEVD, Ile-Glu-Thr-Asp, conjugated to p-nitroanilide (pNA), MADD Mitogen activated kinase activating death domain. MADD interacts with TNF-RI and a colorimetric substrate for Caspase-8-like enzyme activity assay. activates MAP kinase activity (J Biol Chem 1997, 272:12069-12075). IEX-1L An apoptosis inhibitor involved in NF-κB mediated cell survival (Science 1998, MAPKKK5 Mitogen activated protein kinase kinase kinase 5, also known as ASK1, MEKK5, 281:998-1001). induces apoptosis (J Biol Chem 1996, 271:31607 and Science 1997, 275:90-94). I-FLICE Inhibitor of FLICE. I-FLICE is also known as CASH, CLARP, FLIP and FLAME-1 (J Biol Mch2 Mammalian CED-3 homologue 2. Mch2 is another name for Caspase-6 (Cancer Res Chem 1997, 272:17255-17257). 1995, 55:2737-2742). IGIF Interferon-gamma-inducing factor, also known as IL-18, enhances FasL mediated Mch3 Mammalian CED-3 homologue 3. Mch3 is another name for Caspase-7 (Cancer Res cytotoxicity (Nature 1995, 378:88-91). 1995, 55:6045-6052). IκBInhibitor of NF-κB. Mch4 Mammalian CED-3 homologue 4. Mch4 is another name for Caspase-10 (Proc Natl IKKs IkB kinases, including IKKα (IKK1), IKKβ (IKK2), IKKγ (IKK3/IKKAP1/NEMO) (Cell Acad Sci USA 1996, 93:7464-7469). 1997, 91:243-252 and J Exp Med 1999, 189:1839-1845). Mch5 Mammalian CED-3 homologue 5. Mch5 is another name for Caspase-8 (Proc Natl ITA Inhibitor of T-cell apoptosis. ITA is a member of IAP family (DNA Cell Biol Acad Sci USA 1996, 93:14486-14491). 1996,15:981-988). Mch6 Mammalian CED-3 homologue 6. Contains a CARD domain and is also known as I-TRAF A TRAF-interacting protein. I-TRAF is also known as TANK (Proc Natl Acad Sci USA Caspase-9 (J Biol Chem 1996, 271:16720-16724). 1996, 93:8241-6). Mcl-1 Myeloid Cell Leukemia 1. A member of the Bcl-2 family, Mcl-1 is a mitochondrial protein that enhances cell viability under apoptotic conditions. K m-E10 A CARD-containing protein with homology to herpesvirus-2 protein E10, also known KIAP Kidney inhibitor of apoptosis protein. This protein blocks apoptosis induced by the as Bcl-10, c-E10, CARMEN, CLAP and CIPER. (J Biol Chem 1999, 274:10287-10292). overexpression of Bax (Biochem Biophys 2000, 279(3):820-831). MEKK5 Map/Erk kinase kinase 5, also known as ASK1, MAPKKK5, induces apoptosis Ku p70 A 70 KDa subunit of Ku autoantigen. Ku p70 is a component of activated DNA-PK. (Science 1997, 275:90-94). Ku p80 A 80 KDa subunit of Ku autoantigen. Ku p80 is a component of activated DNA-PK. MIAP Mouse inhibitor of apoptosis protein. MIAP is a homologue to human XIAP (Cell 1995, 83:1243-1252). L MICE Mini-ICE, known as Caspase-14 (J Biol Chem 1998,273:29648-29653). MORT1 A death domain containing adaptor that is another name for FADD (J Biol Chem Lamin A A structural protein of the nuclear envelope that assists in maintaining the nuclear 1995, 270:7795-7798). shape (Proc Natl Acad Sci USA 1986, 83:6450-6454). MRIT Mach-Related Inducer of Toxicity. MRIT interacts with Bcl-xL and FLICE and is also Lamin B A structural protein of the nuclear envelope that assists in maintaining the nuclear known as FLIP, CASH, FLAME-1, CLARP, Casper and I-FLICE (Proc Natl Acad Sci USA shape. 1997, 94:11333-11338). LARD Lymphocyte-associated receptor of death. LARD is a receptor for Apo-3L and is also Mtd A member of the Bcl-2 family that contains BH1, BH2, BH3 and BH4 domains and known as DR3, WSL-1, TRAMP and APO-3 (Proc Natl Acad Sci USA 1997, 94:4615- activates apoptosis in the absence of heterodimerization with Bcl-2 and Bcl-XL (J Biol 4619). Chem 1998, 273:8705-8710). LBR A chromatin and lamin binding protein from the inner nuclear membrane. LBR is proteolyzed during the late stages of apoptosis (J Cell Sci 1998, 111:1441-1451). LEHD-AFC A synthetic tetrapeptide LEHD (Leu-Glu-His-Asp) conjugated to 7-amino-4-trifluo- N romethyl coumarin, LEHD-AFC is a fluorogenic substrate for Caspase-1. NAC Protein that contains an NB and CARD domain. NAC interacts with Apaf-1 and LEHD-AMC A synthetic tetrapeptide LEHD (Leu-Glu-His-Asp) conjugated to 7-amino-4-methyl enhances Apaf-1-apoptosome function (J Biol Chem 2001, 276(12):9239-9245). coumarin, LEHD-AMC is a fluorogenic substrate for Caspase-9. NAIP Neuronal apoptosis inhibitory protein. NAIP is a member of the IAP family and LEHD-CHO Leu-Glu-His-Asp-aldehyde. LEHD-CHO is a synthetic Caspase-9 inhibitor. inhibits apoptosis (Cell 1995, 80:167-178). LEHD-pNA A synthetic tetrapeptide LEHD (Leu-Glu-His-Asp) conjugated to p-nitroanilide (pNA), NBK Natural Born Killer. NBK induces apoptosis and is also known as Bik, BP4 and Bip1 a colorimetric substrate for Caspase-9 enzyme activity. (Mol Cell Biol 1996, 16:5857-5864). LEVD-AFC A synthetic tetrapeptide LEVD (Leu-Glu-Val-Asp) conjugated to 7-amino-4-trifluo- NDF Neu differentiation factor, also called neuregulin, induces apoptosis (J Exp. Med romethyl coumarin, LEVD-AFC is a fluorogenic substrate for Caspase-4. 1998, 188:1535-39). LEVD-CHO Leu-Glu-Val-Asp-aldehyde. LEVD-CHO is a synthetic Caspase-4 inhibitor. Nedd2 Neural Precursor Cell Expressed, Developmentally Down-Regulated 2, mouse gene LIGHT A ligand for lymphotoxin b receptor and TR2/HVEM, induces apoptosis (J Clin Invest for Caspase-2. 1998, 102:1142-51). NF-κB Nuclear factor of immunoglobulin k locus in B cells. NF-κB activates transcription of LIT Lymphocyte inhibitor of TRAIL. LIT is also known as TRID, DcR-1 and TRAIL-R3 genes in many tissues. (J Immnuol 1998, 160:3-6). NGF Nerve Growth Factor. LKLF Lung Kruppel-like factor. LKLF is a transcriptional regulator of single-positive T cell NGFR Nerve Growth Factor Receptor (Cell 1986, 47:545-554). quiescence and survival (Science 1997, 277:1986-1990).

e-mail: [email protected] Technical/Customer Services 1-800-242-0607 NIK NF-κB-inducing kinase. It is a serine/threoine kinase which activates IKKα (Proc Natl RIP2 Receptor interacting protein-2, also known as RICK and CARDIAK. It is a NF-κB-acti- Acad Sci USA 1998, 95:3792-7). vating and cell death-inducing kinase (J Bio Chem, 273:16968-16975). NIP-3 Nineteen kD interacting protein-3, an E1B 19K and Bcl-2 binding protein which RIP3 Receptor interacting protein-3. a RIP-like kinase that activates apoptosis and NF-κB. It induces apoptosis (J Biol Chem 1999, 274:7-10). is a potent inducer of apoptosis, capable of selectively binding to large prodomain ini- NIPK Neuronal cell death inducible putative kinase. Involved in programmed cell death tiator caspases. (Curr Biol 1999, 9:539-542 and J Biol Chem 1999, 274:16871-16875) via a pathway that is present in neurons but is absent in non-neurons (Biochem Biophys Res Commun 1999, 258:260-264). S NIX A homolog of the E1B 19K/Bcl-2 binding and pro-apoptotic protein Nip3. Nix and SADS Small accelerator for death signaling. A component of the Fas-DISC complex (Nat Nip3 form a subfamily of pro-apoptotic mitochondrial proteins (J Biol Chem 1999, Med 2001, 7(1):88-93). 274:7-10). SCA-1 SREBP cleavage activity 1. SCA-1 is another name for Caspase-3 (J Biol Chem 1995, Nod1 A CED-4/Apaf-1-like molecule that can regulate both apoptosis and NF-κB activation 270:18044-18050). pathways. Also known as CARD4 (J Biol Chem 1999, 274:14560-14567). SCA-2 SREBP cleavage activity 2. SCA-2 is another name for Caspase-7/Mch3 (Proc Natl Acad Sci USA 1996, 93:5437-5442). O SCC-S2 A TNF-α inducible gene containing sequence homology to FLIP (J Biol Chem 2000, OPG Osteoprotegerin. OPG is a decoy receptor of TRAIL (J Biol Chem 1998, 273:14363- 275(4):2973-2978). 14367). SODD Silencer of death domain. It acts as a silencer of TNF-RI signaling and prevents apop- OX-40 A member of the nerve growth factor family that is expressed on activated lympho- tosis (Science 1999, 283:543-546). cytes. OX-40 is also know as CD134. SREBP-1 Sterol regulatory element binding protein-1. A basic helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene (Cell P 1993, 75:187-197). SREBP-2 Sterol regulatory element binding protein-2. A basic-helix-loop-helix-leucine zipper p53 A tumor suppressor gene product that promotes apoptosis. protein that stimulates transcription by binding to sterol regulatory element (Proc PAAD A protein domain associated with apoptosis, cancer and autoimmune disease (Trends Natl Acad Sci USA 1993, 90:11603-11607). Biochem Sci 2001 26(2):85-87). Survivin A member of the inhibitors of apoptosis protein family. Survivin is anti-apoptosis PAK-1 p21-activated kinase 1. PAK-1 phosphorylates Bad and promotes cell survival (Mol (Nature Med 1997, 3:917-921). Cell Bio 2000, 20(2):453-461). PARP Poly (ADP ribose) polymerase. A DNA repair enzyme cleaved by caspases during apoptosis. T PCD Programmed Cell Death. TACI Transmembrane activator and interacts with CAML. Perforin A protein secreted mainly by cytotoxic T cells or NK cells that punctures a pore on a TANK TRAF family member-associated NF-κB activator. TANK is also known as I-TRAF cell membrane (J Immunol 1989, 143:4267-4274). (Proc Natl Acad Sci USA 1996, 93:11085-11090). PERP An apoptosis associated target of p53 (Genes Dev 2000, 14(6):704-718). THANK TNF homologue that activates apoptosis, and NF-κB, and JNK, a member of TNF PFP Pore-forming protein. PFP is also known as perforin (Immunogenetics 1989, 30:452-457). family (J Biol Chem 274:15978-15981). PI Propidium Iodide. A DNA dye for excluding dead cells. Combined with Annexin-V TIAR RNA binding protein related to TIA-1. Both proteins are effectors of apoptotic cell FITC, PI distinguishes early apoptotic cells from late apoptotic or necrotic cells. death. During Fas-mediated apoptosis, TIAR exhibits rapid translocation from the PKR Double-stranded (ds) RNA-dependent protein kinase. It is a key mediator of antiviral nucleus to the cytoplasm. effects of interferon (IFN) and an active player in apoptosis induced by different stimuli. TL1 A TNF-like cytokine, induces apoptosis in endothelial cells (J Biol Chem 1999, (Mol Cell Biol 1999, 19:4653-4663). 274:1479-1486). PS Phosphatidylserine. PS is a lipid that translocates to the cell surface when cells go TL2 A TNF-like cytokine, also known as TRAIL (Gene 1997, 204:35-46). into apoptosis. PS may be detected by Annexin-V. TLR2 Toll-like receptor 2. A signal for apoptosis through MyD88 via FADD and Caspase-8 (EMBO J 2000, 19(13):3325-3336). R TNF Tumor necrosis factor. TNF-RI Tumor necrosis factor receptor I. A 55 kDa protein (p55) that is also known as RAIDD RIP associated Ich-1/CED homologous protein with death domain. RAIDD is a death CD120a. adaptor molecule (Nature 1997, 385:86-89). TNF-RII Tumor necrosis factor receptor II. A 75 kDa protein (p75) that is also known as RANK Receptor activates NF-κB. RANK interacts with tumor necrosis factor receptor-asso- CD120b. ciated factors, NF-κB and Jak (c-jun n-terminal kinase) (J Biol Chem 1998, Toso A cell surface specific regulator of Fas-induced apoptosis in T cells (Immunity 1998: 273:20551-20555). 8:461-471). RANKL RANK ligand, also know as TRANCE or osteoprotegerin ligand. It is a member of TNF TR1 TNF receptor related 1, also known as osteoprotegrin (OPG) (Gene 1997,204:35-46). family (Nature 1997, 390:175-179). TR2 TNF receptor related 2, also known as HVEM (J Immunol 1998, 161:1786-94). RICK A protein kinase containing a caspase recruitment domain, also known as RIP2 and TR2L TR2L protein inhibited TNF cytotoxic response (Biochem Biophys Res Commun CARDIAK. RICK interacts with CLARP and regulates CD95-mediated apoptosis (J Biol 1996, 227:266-272). Chem 1998, 273:12296-12300). TR3 TNF receptor related 3, also known as DR3/WSL-1/Apo-3/TRAMP/LARD (Gene 1997, RIP Receptor interacting protein. A Fas-binding protein containing death domain that is 204:35-46). crucial for TNF-RI mediated NF-κB activation (Cell 1995, 81:513-523). e-mail: [email protected] Technical/Customer Services 1-800-242-0607 TRADD TNF Receptor I associated death domain. Over-expression of TRADD leads to two major VEID-AFC A synthetic tetrapeptide, Val-Glu-Ile-Asp, conjugated to 7-amino-4- trifluoromethyl TNF-induced responses, apoptosis and activation of NF-κB (Cell 1995, 81:495-504). coumarin. VEID-AFC is a fluorometric substrate for Caspase-6 enzyme activity assay. Traf-1 TNF Receptor Associated factor-1. A signal transducer associated with the cytoplas- VEID-AMC Val-Glu-Ile-Asp-7-amino-4-methyl coumarin. VEID-AMC is a fluorogenic substrate mic domain of TNF receptor II (Cell 1994, 78:681-692). for Caspase-6. Traf-2 TNF Receptor Associated factor-2. Mediates CD30-induced NF-κB activation (Proc VEID-CHO Val-Glu-Ile-Asp-aldehyde. VEID-CHO is a synthetic Caspase-6 inhibitor. Natl Acad Sci USA 1996, 93:14053-14058). VEID-FMK Val-Glu-Ile-Asp-O-methyl-fluoromethylketone. VEID-FMK is a synthetic Caspase-6 Traf-3 TNF Receptor Associated factor-3. Interacts with CD30 cytoplasmic domain (Proc inhibitor. Natl Acad Sci USA 1996, 93:14053-14058). VEID-pNA A synthetic tetrapeptide Val-Glu-Ile-Asp conjugated to p-nitroanilide (pNA). VEID- Traf-4 TNF Receptor Associated factor-4. Traf-4 interacts with the cytosolic domain of the pNA is a colorimetric substrate for Caspase-6 like enzyme activity assay. lymphotoxin receptor (Am J Pathol 1998, 152:1549-1561). Traf-5 TNF Receptor Associated factor-5. Traf-5 is a member of the TRAF family and medi- W ates CD40 signaling (Proc Natl Acad Sci USA 1996, 93:9437-9442). WSL-1 A death-domain-containing receptor that mediates apoptosis. WSL is also known as Traf-6 TNF Receptor Associated factor-6. Traf-6 mediates CD40 signaling (J Biol Chem APO3, DR3, TRAMP and LARD (Nature 1996, 384:372-375). 1996, 271:28745-28748). TRAIL Tumor necrosis factor (TNF)-related apoptosis-inducing ligand. TRAIL is also known as Apo-2L and is a member of the TNF family that induces apoptosis (Immunity 1995, X 3:673-682). XIAP X-linked inhibitor of apoptosis protein. XIAP, also known as HILP, is a member of IAP TRAIL-R1 TRAIL-receptor 1. TRAIL-R1 is also known as DR4 and AOP-2 (Immunity 1997, family that inhibits apoptosis (EMBO 1998, 17:2215-2223). 7:831-836). TRAIL-R2 TRAIL-receptor 2. TRAIL-R2 is also known as DR-5 (Immunity 1997, 7:821-830). Y TRAIL-R3 TRAIL-receptor 3. TRAIL-R3 is also known as DcR1, TRID and LIT (J Exp Med 1997, Yama Another name for Caspase-3 (J Biol Chem 1994, 269:30761-30764). 186:1165-1170). YAVD-AFC A synthetic tetrapeptide YVAD (Tyr-Val-Ala-Asp) conjugated to 7-amino-4-trifluo- TRAIL-R4 TRAIL-receptor 4. TRAIL-R4 is also known as DcR2 (Curr Biol 1997, 7:1003-1006). romethyl coumarin, YAVD-AFC is a fluorogenic substrate for Caspase-1. TRAMP TNF receptor apoptosis-mediating protein. TRAMP is an apoptosis-mediating recep- YAVD-AMC A synthetic tetrapeptide YVAD (Tyr-Val-Ala-Asp) conjugated to 7-amino-4-methyl tor with homology to TNF-RI and Fas. TRAMP is also known as DR3, APO-3, WSL, coumarin, YAVD-AMC is a fluorogenic substrate for Caspase-1. and LARD (Immunity 1997, 6:79-88). YAVD-CHO Tyr-Val-Ala-Asp-aldehyde. YAVD-CHO is a synthetic Caspase-1 inhibitor. TRANCE TNF-related activation-induced cytokine. TRANCE is a member of the TNF family YVAD Amino acid residues (Tyr-Val-Ala-Asp) of Caspase-1 cleavage site within interleukin-1β. and expressed predominantly in T cells (J Biol Chem 1997, 272:25190-25194). TRAP Tumor necrosis factor-related activation protein. A member of the TNF superfamily YVAD-pNA A synthetic tetrapeptide YVAD (Tyr-Val-Ala-Asp) conjugated to p-nitroanilide (pNA), that is also known as CD40 ligand and CD154 (Eur J Immunol 1992, 22:3191-3194). a colorimetric substrate for Caspase-1 enzyme activity. TRICK2 Trail receptor inducer of cell killing 2. An alternatively spliced receptor that trans- duces the cytotoxic signal from TRAIL (Curr Biol 1997, 7:693-696). TRID TRAIL receptor without an intracellular domain. TRID is an antagonist decoy recep- Z tor and is also known as DcR1, TRAIL-R3 and LIT (Science 1997, 277:815-818). ZIP kinase Zipper-interacting protein kinase, a serine/threonine kinase that mediates apoptosis TRIP TRAF interacting protein. A component of TNFR- and CD30-TRAF signaling complexes (Mol Cell Biol 1998, 18:1642-1651). that inhibits TRAF2-mediated NF-κB activation (J Exp Med 1997, 185:1275-1285). zVAD-FMK Benzyloxy-valine-alanine-aspartate-O-methyl-fluoromethylketone. zVAD-FMK is a TRUNDD Trail receptor with a truncated death domain that plays an inhibitory role in apoptosis synthetic caspase inhibitor. (FEBS Lett 1998, 424:41-45). TUNEL Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling. A method for detecting apoptotic cells based on the DNA fragmentation during apoptosis. TWEAK A secreted ligand in the TNF family that weakly induces apoptosis (J Biol Chem 1997, 272:32401-32410). TX TX protease. TX is another name for Caspase-4 (EMBO J 1995, 14:1914-1922). TY TY protease. TY is another name for Caspase-5 (J Biol Chem 1995, 270:15870- 15876). V VDVAD-AFC A synthetic peptide VDVAD(Val-Asp-Val-Ala-Asp) conjugated to 7-amino-4-trifluoromethyl coumarin, VDVAD-AFC is a fluorogenic substrate for Caspase-2. VDVAD-pNA A synthetic peptide VDVAD(Val-Asp-Val-Ala-Asp) to p-nitroanilide (pNA), a colorimetric substrate for Caspase-2 enzyme activity. VEID Val-Glu-Ile-Asp, amino acid residues which correspond to one of the Caspase-6 cleavage sites of lamin A.

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