Cell Biology - Assays Kits Apoptosis For your research Interchim offers a comprehensive line of products : Technical tip ! Membrane assays (AnnexinV, AppolerCentage) ! Caspase assays Apoptosis is a programmed, cell-autonomous death process, known to impact every stage of ! Mitochondria assays life of multicellular eukaryotes. It is involved in a variety of physiological and pathological events, ranging from normal development (embryo Method Advantages Disadvantages growth, postnatal organ modeling and cell Apoptosis membrane alteration : Dye Single apoptotic cell (IHC). Quick and Limited information available on cell turnover) to diseases and disorders (cancer, (uptake bioassay: APOPercentage). easy to use. Assay can also be membrane composition. organ failure and ageing, and quantified, using a digital camera or a neurodegenerative diseases). During microplate colorimeter/fluorimeter. apoptosis, the caspases execute the Necrotic cells cannot retain the dye. disassembly of cellular components by Several hundred assays can be proteolytic cleavage of a variety of substrates, such as poly-(ADP ribose) polymerase (PARP), performed in one day. DNA-dependent protein kinase (DNA-PK), Apoptosis membrane alteration: Sensitivity Single cell (FCM, IHF). Does not discriminate between apoptotic topoisomerases, and protein kinase C (PKC). (Annexin V binding) Confirms the occurrence of and necrotic cells. At least ten caspases have been discovered. phosphatidylserine flippase in Some of these caspases identify and cleave a apoptotic cells, and the activity of specific peptide substrate, while others initiator caspases. recognize the same peptide substrate. Fluorescence Activated Cell Sorter Good option for suspension cells. The equipment is designed for cell (FACS). counting and cell sorting. Excessive physical force, to remove cell clumps, can damage cells. Lower Sensitivity (>100 Selection guide apoptotic cells to discern cluster in a sample scan. Apoptotic proteases It is possible to select for individual Lysis or fixation of cells (except for (caspases assays) initiator caspases or execution CytoxiLux)Caspase activation does not caspases. Sensitivity : Single cell necessarily imply that apoptosis will (with FCM) (IEA or IFA: occur. >usually 1 x 105 cells) Detection and measurement of specific caspase activity. Enzymatic end labelling of DNA Can be completed within 3 hours. Fixation of cells. Subject to false positives strand breaks (TdT TUNEL: TACS) Fluorescence microscopy or FACS. from necrotic cells and risk of high Sensitivity. Single cell background from some viable cells. Collection times can be critical, early collection and DNA fragmentation may not yet be extensive, or later and DNA Cell Biology fragmentation can be excessive. DNA fragmentation Photographic evidence of large DNA Can be difficult to produce a nucleosome (BET/Electrophoresis : Commet Assay) fragmentsSingle cell ladder as further DNAfragmentation can (with Comet Assay) occur during preparation. Necrotic cells also generate DNA fragments Sensitivity. 1 x 106 cells Apoptosis Cell and Tissue Control Slides Following control slides will help developing familiarity with the methods of in situ apoptosis labeling and to determine if reagents are working optimally. E.156 Description Cat.# Qty Cell Culture Control Slides Q69270 2 u Q69271 5 u Tissue Control Slides Q69290 2 u Q69291 5 u See also Antibodies Research Area #3) ( Apoptosis, and related Abs) and Last minute Abs (moitochondrial research). See also M-PCR Kits Tel 33 (0)4 70 03 88 55 !! Hot line 33 (0)4 70 03 73 06 Fax 33 (0)4 70 03 82 60 Cell Biology - Assays Kits Apoptosis Membrane apoptosis events Labeled Annexin V Exposure of phosphatidyl serine One of the first identifiable events during apoptosis is the «flipping» of phosphatidyl serine (PS) on the cell membrane, resulting in exposure of PS on the cell surface. The anticoagulant annexin V binds specifically to PS in a calcium-dependant event. PS translocation to the cell surface precedes nuclear breakdown, DNA fragmentation, and the appearance of most apoptosis-associated molecules, making annexin V binding a marker of apoptosis early-stage. Annexin V is available conjugated with following labels by an optimized chemistry to ensure proper PS binfing. Description Cat.# Qty Annexin V - FluoProbes 488 FP-BH4140 500 µl for confocal microscopy 494/519 nm Annexin V - FluoProbes 488 FP-BH9390 100 tests for flow cytometry 494/519 nm Annexin V - FITC FP-M19652 250 µl 494/518 nm Annexin V - R-phycoerythrin (RPE) FP-AH191A 50 tests 496, 546, 565/578 nm Annexin V - Allo-phycocyanin (APC) FP-AK194A 50 tests 650/660 nm Annexin V - X - Biotin FP-M1969A 500 µe Related products : Cell Biology 30 fluorescents streptavidins can be chosen. see page A350 Anti - Annexin V - FITC R51380 100 tests 494/518 nm Annexin V, 10x binding buffer BU2080 1.7 ml Comparison of the performances of Annexin V- FluoProbes® 448 by flow cytometry and Annexin V-FITC. The Jurkat cells are incubated with anti-Fas antibody for 3 hours to activate apoptosis. The activated cell suspension is then mixed with Annexin V- FluoProbes® 488 or Annexin V-FITC and further incubated for 15 minutes. E.157 Figure 2 shows the fluorescence intensities histograms of the cells incubated with annexin V-FluoProbes® 488. Table lists the Mean Fluorescence Intensities of the viable cells (M1) and the apoptotic cells (M2) and the ratio of M2/M1 with annexin V- FluoProbes® 488 or annexin V-FITC. Performance of Annexin V-FITC and Annexin V-Fluoprobes Figure 2 : Histogram of the cells incubated with 488 with Confocal Scanning Laser Microscopy. annexin V-FP488 The Annexin V-Fluoprobes 488 is superior to the FITC probe, both in quantum yield and in resistance to bleaching. Associated product : Prodium iodie Iodide FP-36774A 10 ml see page E123. 7.AAD FP-132303 1 mg see page E126 used for dead cell identification. e-mail [email protected] ! Visit our website : www.interchim.com Cell Biology - Assays Kits Apoptosis TACS™ Annexin V Kits detection of apoptotic cells Features : ! Fast, sensitive detection of apoptotic cells. ! Includes propidium iodide for discriminating apoptotic and necrotic (or late apoptotic) cells. ! Annexin V-Biotin provides flexibility in fluorophore choice. TACSTM Annexin V Kits allow rapid, specific, and quantitative identification of apoptosis in individual cells. They use annexin V conjugated to either FITC or to biotin for the detection of cell surface changes during apoptosis. The annexin V conjugates are supplied with an optimized Binding Buffer and propidium iodide. Propidium iodide may be used on unfixed samples to determine the population of cells that have lost membrane integrity, an indication of late apoptosis or necrosis. Description Cat.# Qty Flow Cytometry analysis of WEHI 7.1 cells labeled with Annexin V-Biotin and detected by streptavidin-FITC. TACS™ Annexin V Kits 372810 100 tests WEHI 7.1 cells treated with 25 µm etoposide for two hours 372811 250 tests with an overnight recovery produce a peak approximately 3 log of 10 in the fluorescence channel 1 (FL1) (samples 2 Related products : and 3). Healthy WEHI 7.1 cells produce a peak less than log 101 in the FL1 channel which is similar to unlabeled Several complementatry probes are described in section E : cells (samples 4.5 and 1 respectively). Analysis was tested using two different populations of cells (samples 1,2,4 and Description Cat.# Qty 3,5 respectively). PI see page E123 FP-36774A 10 ml @ 1 mg/ml 7-AAD is used for dead cell identification See page E126 FP-132303 1 mg APOPercentage™ APOPTOSIS Assay The APOPercentage™ Assay is a detection and MEASUREMENT system to monitor the incidence of apoptosis in mammalian anchorage-dependent cells during in vitro culture. Apoptotic cells take up the APOPercentage dye, as shown in the pictures below. Following dye uptake, the level of apoptosis can be quantified colorimetrically, fluorometrically or by analytical digital photomicroscopy (ADP). Cell Biology 2 hr 4hr 6hr ! Assay time : 1 hour Digital images of CHO cells treated with 10 mM ! Assay sensitivity : a single apoptotic cell. cycloheximide, to induce apoptosis after 2, 4 and 6h. ! Compatible with CDD camera znd microplate readers Description Cat.# Qty APOPercentage™ Apoptosis Assay kits : Trial size Kit (3 x 96 wells; microwell format) R47770 250 tests E.158 Standard Assay Kit (6 x 96 wells; microwell format) R47771 500 tests Components of the # R47770 kit : APOPercentage Dye reagent (5 ml) in phosphate buffered saline (sterile vial) APOPercentage Dye Reference Standard (10 µM/10 ml), (sterile vial) APOPercentage Dye Release reagent (150 ml) Gelatin matrix forming solution (0.4%/100 ml / sterile) Tel 33 (0)4 70 03 88 55 !! Hot line 33 (0)4 70 03 73 06 Fax 33 (0)4 70 03 82 60 Cell Biology - Assays Kits Apoptosis Biochemicals for apoptosis research Caspase inhibitor kit Features : ! Synthetic peptides coupled to fluoromethylketone (FMK) which irreversibly binds the caspase. ! High concentrations of inhibitor to reduce risks of DMSO toxicity. ! Each kit is provided with a negative control and a general caspase inhibitor. ! Application range from 0.2 to 200 µM. Description Cat.# Qty Caspase 3 Inhibitor Kit Q70200 20 µl Caspase 8 Inhibitor Kit Q70220 20 µl Caspase 9 Inhibitor Kit Q70240 20 µl Caspase 3,8,9 Inhibitor Kit Q70180 20 µl Each Apoptosis Inducers Valinomycin is used to induce mitochondrial potential disruption. Etoposide is a DNA Schematic representation of the pathways of synthesis inhibitor that induces double stranded and single stranded DNA breaks. caspaces activation and where individual inhibitors Staurosporine is a phospholipid/calcium-dependent protein kinase inhibitor that prevents will have an effect. ATP binding. Description Cat.# Qty Technical tip Valinomycin FP-09246B 25 mg 092460 100 µl Applications : Etoposide FX8650 100 µl Caspase inhibitors are useful tools, added to Cell Biology Staurosporine 74146D 100 µg cell culture or cell-free extracts, for studying 74146E 1 mg relationships between caspases and other FX8660 100 µl factors involved in apoptosis. Caspase inhibitors inactivate specific caspases allowing study of their impact on pathways of interest, or of other caspases or apoptotic events.