BD ApoAlert™ Caspase Fluorescent Assay Kits User Manual
Contents
1. Brown D G Langlais C amp Cohen G M 1999 Caspase activation involves the formation of the aposome a large 700 kDa caspase activating complex J Biol Chem 274 22686 22692 Cohen G M 1997 Caspases the executioners of apoptosis Biochem J 326 1 16 Fernandes Alnemri T Litwack G amp Alnemri E S 1994 CPP32 a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced 3 and mammalian interleukin 1 beta converting enzyme J Biol Chem 269 30761 30764 Fraser A amp Evan G 1996 A license to kill Cell 85 6 781 784 Green D R amp Reed J C 1998 Mitochondria and apoptosis Science 281 1309 1312 Hu Y Benedict M A Ding L amp Nunez G 1999 Role of cytochrome c and dATP ATP hydrolysis in Apaf 1 mediated caspase 9 activation and apoptosis EMBO J 18 3586 3595 Muzio M Salvesen G S amp Dixit V M 1997 FLICE induced apoptosis in a cell free system J Biol Chem 272 2952 2956 Nicholson D W 1996 ICE CED3 like proteases as therapeutic targets for the control of inappropriate apoptosis Nature Biotechnol 14 297 301 Nicholson D W amp Thornberry N A 1997 Caspases killer proteases Trends Biochem Sci 22 299 306 Porter A G amp Janicke R U 1999 Emerging roles of caspase 3 in apoptosis Cell Death Diff 6 99 104 Srinivasula S M Ahmad M Fernandes Alnemri T Litwack G amp Alnemri E S 1996 Molecular2. Transfer supernatants to new microcentrifuge tubes Note At this point samples may be assayed immediately or frozen for assay at a later time 6 Optional In the absence of a positive control reaction with purified Caspase 8 incubate an induced sample with Caspase 8 Inhibitor before adding substrate This reaction verifies that the signal detected by the kit is due to protease activity Add 50 ul of 2X Reaction Buffer DTT Mix Section V A and 1 ul of Caspase 8 Inhibitor IETD fmk to 50 ul of supernatant from a sample obtained in Step 5 Incubate on ice for 30 min together with the other samples Proceed to Step 7 where you will add reaction buffer to the remaining samples 7 Add 50 ul of 2X Reaction Buffer DTT Mix Section V A to each reaction If you are using the Caspase 8 Inhibitor as a negative control add 1 ul DMSO per 50 ul of 2X Reaction Buffer to samples without inhibitor to ensure that all samples are tested under similar conditions 8 Add 5 wl of the 1 mM Caspase 8 Substrate IETD AFC 50 uM final conc to each tube Incubate at 37 C for 1 hr or up to 3 hours maximum in a water bath Set up a parallel control reaction that does not contain conjugated substrate 9 Read in a fluorometer with a 400 nm excitation filter and 505 nm emission filter For plate reading transfer samples to a 96 well plate For quantification of protease activity proceed to Section VII A Protocol PT3191 1 www bdbiosciences com B
3. ordering of the Fas apoptotic pathway the Fas APO 1 protease MCH 5 is a CrmA inhibitable protease that activates multiple Ced 3 ICE like cysteine proteases Proc Natl Acad Sci USA 93 14486 14491 Thornberry N A amp Littlewood Y 1998 Caspases Enemies Within Science 281 1312 1316 Zou H Li Y Liu X amp Wang X 1999 An APAF 1 cytochrome c multimeric complex is a functional apoptosome that activates procaspase 9 J Biol Chem 274 1 1549 11556 Protocol PT3191 1 www bdbiosciences com BD Biosciences Clontech Version PR26395 13 BD ApoAlert Caspase Fluorescent Assay Kits User Manual IX Related Products For a complete listing of all BD Biosciences Clontech products please visit www bdbiosciences com Apoptosis Inducing Reagent Human TNF a BD ApoAlert Apoptosis Inhibiting Reagents Caspase 8 Inhibitor IETD fmk e Caspase 3 Inhibitor DEVD CHO e Caspase 3 Inhibitor DEVD fmk e Caspase 1 Inhibitor YVAD cmk e Caspase Inhibitor VAD fmk BD ApoAlert Apoptosis Assay Kits e Caspase 9 6 Fluorescent Assay Kit e Caspase 8 Assay Kits Fluorescent Colorimetric e Caspase 3 Assay Kits Fluorescent Colorimetric Mitochondrial Membrane Sensor Kit DNA Fragmentation Assay Kit e Annexin V Apoptosis Kit e Cell Fractionation Kit BD Biosciences Clontech www bdbiosciences com 14 8157 1 8174 1 8170 1 8172 1 8171 1 8173 1 K2015 1 2 K2028 1 2 K2029 1 2 K2026 1 2
4. apoptotic sample with an uninduced control allows determination of the fold increase in protease activity Units of protease activity can also be quantitated accurately and repro ducibly using a standard curve established with the appropriate free fluorescent molecule Section VII Cells You may perform the assay in 0 5 1 5 ml reaction tubes or 96 well plates Use at least 1 x 10 cells per sample Using fewer cells may reduce the observed increase in protease activity BD Biosciences Clontech www bdbiosciences com Protocol PT3191 1 Version PR26395 BD ApoAlert Caspase Fluorescent Assay Kits User Manual IV BD ApoAlert Caspase 3 Assay Protocol continued B Assay Procedure 1 A Induce apoptosis in cells by desired method Remember to incubate a concurrent control culture without induction Setup duplicate cell plates for the following samples induced uninduced negative control induced plus inhibitor Step 6 optional and in duced without substrate Step 8 Count cells and centrifuge 1 x 10 cells at 400 x g for 5 min Note After removing the supernatant you may freeze the cell pellets at 70 C and assay at a later time Resuspend cells in 50 ul of chilled Cell Lysis Buffer Incubate cells on ice for 10 min Centrifuge cell lysates in a microcentrifuge at maximum speed for 10 min at 4 C to precipitate cellular debris Transfer the supernatants to new microcentrifuge tubes Not
5. are part of alarge family of cysteine proteases that mediate programmed cell death apoptosis Upon activation they disable cellular homeostatic and repair processes and cleave important structural components in the cell causing morphological and functional changes to cells undergoing apoptosis Background Caspases cleave a variety of cellular substrates after aspartic acid residues a characteristic that is central to their role in mammalian apoptosis Caspases are synthesized in the cytosol of mammalian cells as inactive zymogens which become active through intracellular caspase cascades Cohen 1997 The BD ApoAlert Caspase Fluorescent Assay Kits allow you to detect the activity of a specific caspase caspase 3 caspase 8 or caspase 9 6 which becomes active at different stages and or different pathways of the apoptotic process Caspase 3 is an active cell death protease involved in the execution phase of apoptosis during which cells undergo morphological changes such as DNA fragmentation chromatin condensation and apoptotic body formation Porter amp Janicke 1999 Zou et al 1999 Caspase 3 is activated in response to serum withdrawal activation of Fas treatment with radiation and pharmacological agents Zou et al 1999 as well as to other upstream caspases such as caspase 8 and caspase 9 Caspase 8 the most upstream caspase in the CD95 apoptotic pathway is activated by the signaling pathways for CD95 Fas and tiss
6. BD ApoAlert Caspase Fluorescent Assay Kits User Manual Cat No K2015 1 2 K2026 1 2 K2028 1 2 PT3191 1 PR26395 Published 08 22 2002 BD ApoAlert Caspase Fluorescent Assay Kits User Manual Table of Contents Vi Vil VIII Introduction List of Components Additional Materials Required Caspase 3 Assay Protocol A General Considerations B Assay Procedure Caspase 8 Assay Protocol A General Considerations B Assay Procedure Caspase 9 6 Assay Protocol A General Considerations B Assay Procedure Quantification of protease activity References Related Products Notice to Purchaser This product is intended to be used for research purposes only It is not to be used for drug or diagnostic purposes nor is it intended for human use BD Biosciences Clontech products may not be resold modified for resale or used to manufacture commercial products without written approval of BD Biosciences Clontech BD ApoAlert is a trademark of Becton Dickinson and Company 2002 Becton Dickinson and Company BD Biosciences Clontech 2 www bdbiosciences com DOAN DOA a Ww ee ae O ce ee S A O N OOO Protocol PT3191 1 Version PR26395 BD ApoAlert Caspase Fluorescent Assay Kits User Manual I Introduction The BD ApoAlert Caspase Fluorescent Assay Kits provide asimple means for detecting the activity of key caspases in mammalian cells caspase 3 caspase 8 and caspase 9 These caspases
7. D Biosciences Clontech Version PR26395 9 BD ApoAlert Caspase Fluorescent Assay Kits User Manual Vi BD ApoAlert Caspase 9 6 Assay Protocol PLEASE READ ENTIRE PROTOCOL BEFORE BEGINNING A General Considerations A relatively high concentration of DTT 10 mM is required for full activity of the enzymes Ensure that DTT is added to the Reaction Buffer when the assay is performed Otherwise unexpected low caspase activity will occur Turbidity or particulate materials in samples can decrease assay precision Reagents Aliquot a sufficient volume of 2X Reaction Buffer 50 ul assay for the number of assays you will perform Immediately before use add DTT to the aliquotted 2X Reaction Buffer add 10 ul of 1 M DTT stock per 1 ml of 2X Reaction Buffer Do not add DTT to stock 2X Reaction Buffer Turbidity or particulate materials in samples can decrease assay preci sion Ensure that all buffers are completely in solution before use Controls We recommend performing three control reactions 1 a negative control on uninduced cells Step VI B 1 2 a control on induced cells treated with Caspase 9 6 Inhibitor Step VI B 6 3 a positive control for Caspase 9 induction You may treat your own cells with a known inducer At BD Biosciences Clontech we use Staurosporine 500 nM for 4 hr or human anti Fas monoclonal antibody clone CH 11 500 ng ml final concentration for 4 12 hr Anti Fas clone DX2 is not recommende
8. K2027 1 2 K2017 1 K2024 1 2 K2025 1 2 K2016 1 Protocol PT3191 1 Version PR26395 BD ApoAlert Caspase Fluorescent Assay Kits User Manual Notes Protocol PT3191 1 www bdbiosciences com BD Biosciences Clontech Version PR26395 15
9. d for use with this kit Comparing the emission of an apoptotic sample with an uninduced control allows determination of the fold increase in protease activity Cells You may perform the assay in 0 5 1 5 ml reaction tubes or 96 well plates Use at least 1 x 10 cells per sample Fewer cells may reduce the observed increase in protease activity BD Biosciences Clontech www bdbiosciences com Protocol PT3191 1 10 Version PR26395 BD ApoAlert Caspase Fluorescent Assay Kits User Manual VI BD ApoAlert Caspase 9 6 Assay Protocol continued B Assay Procedure 1 Induce apoptosis in cells by desired method Remember to incubate a concurrent control culture without induction Set up duplicate cell plates for the following samples induced uninduced negative control induced plus inhibitor Step 6 optional and in duced without substrate Step 8 2 Count cells and centrifuge 1 x 10 cells at 400 x g for 5 min Note After removing the supernatant you may freeze the cell pellets at 70 C and assay at a later time 3 Resuspend cells in 50 ul of chilled Cell Lysis Buffer Incubate cells on ice for 10 min 5 Centrifuge cell lysates in a microcentrifuge at maximum speed for 10 min at 4 C to precipitate cellular debris Transfer supernatants to new microcentrifuge tubes Note At this point samples may be assayed immediately or frozen for assay at a later time 6 Optional In the absence of a positive con
10. e At this point samples may be assayed immediately or frozen for assay at a later time Optional In the absence of a positive control reaction with purified Caspase 3 incubate an induced sample with Caspase 3 Inhibitor before adding substrate This reaction verifies that the signal detected by the kit is due to protease activity Add 50 ul of 2X Reaction Buffer DTT Mix Section IV A and 1 ul of Caspase 3 Inhibitor DEVD CHO to 50 ul of supernatant from a sample obtained in Step 5 Incubate on ice for 30 min together with the other samples Proceed to Step 7 where you will add reaction buffer to the remaining samples Add 50 ul of 2X Reaction Buffer DTT Mix Section IV A to each reaction If you are using the Caspase 3 Inhibitor as a negative control add 1 ul DMSO per 50 ul of 2X Reaction Buffer to samples without inhibitor to ensure that all samples are tested under similar conditions Add 5 ul of 1 mM Caspase 3 Substrate DEVD AFC 50 uM final conc to each tube Incubate at 37 C for 1 hr or up to 3 hours maximum in a water bath Set up a parallel control reaction that does not contain conjugated substrate Read in a fluorometer with a 400 nm excitation filter and 505 nm emission filter For plate reading transfer samples to a 96 well plate For quantification of protease activity proceed to Section VII Protocol PT3191 1 www bdbiosciences com BD Biosciences Clontech Version PR26395 7 BD ApoAlert Cas
11. ion Curve 1 2 Label four 0 5 ml microcentrifuge tubes as shown in the diagram below Prepare 4 uM of AFC solution in 1X Cell Lysis Buffer Add 380 ul 1 X Cell Lysis Buffer to tube 1 then add 20 ul of AFC stock solution provided Mix well To the remaining tubes add 200 ul 1X Cell Lysis Buffer and perform a serial dilution From tube 1 add 200 ul into tube 2 and mix well Then from tube 2 add 200 ul into tube 3 and mix well Do the same for tube 4 200ul 200ul 200u 2004 ARES eG Ne v 2 Add 380 wl Cell Lysis Add 200 ul Cell Lysis Buffer Buffer tubes 2 4 Final AFC 4uM 2uM 1uM 0 5uM Concentration Measure the samples in a fluorometer 400 nm Ex 505 nm Em using cell lysis buffer without AFC for background calibration Prepare a calibration curve with x uM AFC and y fluorescence units FU Use the slope AFU AuM AFC of the curve to calculate caspase activity with the following formula Caspase activity AFU hr x 1 curve slope AFU hr the difference in FU between an uninduced control and an induced sample Note Similiar quantification can be performed for the Caspase 9 6 assay using AMC Section III Measure the samples in a fluorometer set at 380 nm Ex 460 nm Em BD Biosciences Clontech www bdbiosciences com Protocol PT3191 1 12 Version PR26395 BD ApoAlert Caspase Fluorescent Assay Kits User Manual Vill References Cain K
12. is not recommended for use with this kit Comparing the emission of an apoptotic sample with an uninduced control allows determination of the fold increase in protease activity Units of protease activity can also be quantitated accurately and repro ducible using a standard curve established with the appropriate free fluorescent molecule Section VII Cells You may perform the assay in 0 5 1 5 ml reaction tubes or 96 well plates Use at least 1 x 10 cells per sample Using fewer cells may reduce the observed increase in protease activity BD Biosciences Clontech www bdbiosciences com Protocol PT3191 1 Version PR26395 BD ApoAlert Caspase Fluorescent Assay Kits User Manual V BD ApoAlert Caspase 8 Assay Protocol continued B Assay Procedure 1 Induce apoptosis in cells by desired method Remember to incubate a concurrent control culture without induction Set up duplicate cell plates for the following samples induced uninduced negative control induced plus inhibitor Step 6 optional and in duced without substrate Step 8 2 Count cells and centrifuge 1 x 10 cells at 400 x g for 5 min Note After removing the supernatant you may freeze the cell pellets at 70 C and assay at a later time 3 Resuspend cells in 50 ul of chilled Cell Lysis Buffer Incubate cells on ice for 10 min 5 Centrifuge cell lysates in a microcentrifuge at maximum speed for 10 min at 4 C to precipitate cellular debris
13. pase Fluorescent Assay Kits User Manual V BD ApoAlert Caspase 8 Assay Protocol PLEASE READ ENTIRE PROTOCOL BEFORE BEGINNING A General Considerations A relatively high concentration of DTT 10 mM is required for full activity of the enzymes Ensure that DTT is added to the Reaction Buffer when the assay is performed Otherwise unexpected low caspase activity will occur Turbidity or particulate materials in samples can decrease assay precision Reagents Aliquot a sufficient volume of 2X Reaction Buffer 50 ul assay for the number of assays you will perform Immediately before use add DTT to the aliquotted 2X Reaction Buffer add 10 ul of 1 M DTT stock per 1 ml of 2X Reaction Buffer Do not add DTT to stock 2X Reaction Buffer Turbidity or particulate materials in samples can decrease assay preci sion Ensure that all buffers are completely in solution before use You may need to heat free AFC to dissolve precipitates that can form during cold storage Controls We recommend performing three control reactions 1 a negative control on uninduced cells Step V B 1 2 a control on induced cells treated with Caspase 8 Inhibitor Step V B 6 3 a positive control for Caspase 8 induction You may treat your own cells with a known inducer At BD Biosciences Clontech we use Staurosporine 500 nM for 4 hr or human anti Fas monoclonal antibody clone CH 11 500 ng ml final concentration for 4 12 hr Anti Fas clone DX2
14. resces yellow green A 505 nm Similarly the Caspase 9 6 Fluorescent Assay Kit detects the emission shift of 7 amino 4 methyl coumarin AMC The LEHD AMC conjugate emits in the UV range Amax 380 nm however free AMC fluoresces blue green at 440 nm upon liberation by caspase 9 6 Each Caspase Fluorescent Assay Kit includes a specific synthetic caspase inhibitor When you are assaying whole cell lysates the inhibitor can be used as a negative control Inhibitors are also available separately for investigating the overall roles of proteases in the apoptotic process See Related Products Section IX for ordering information Induction of apoptosis in cells Protease activation lay Caspase 3 Caspase 8 Caspase 9 6 DEVD AFC JETD AFC LEHD AMC DEVD J IETD SULA Wz W Z Er al TW TTS fluorometric detection Figure 1 Detecting protease activity using BD ApoAlert Caspase Fluorescent Assay Kits Fluorometric detection for caspase 3 and caspase 8 is performed using a 400 nm excitation filter and 505 nm emission filter To detect caspase 9 6 fluorometric detection is performed using a 380 nm excitation filter and 460 nm emission filter BD Biosciences Clontech www bdbiosciences com Protocol PT3191 1 Version PR26395 BD ApoAlert Caspase Fluorescent Assay Kits User Manual ll List of Components Store Cell Lysis Buffer and 2X Reaction Buffer at 4 C after opening Free AFC may be stored at 4 C after first u
15. se Store substrate inhibitor and DTT at 20 C Caspase 3 Fluorescent Assay Kits 25 assays 100 assays K2026 1 K2026 2 32 ml 125 ml Cell Lysis Buffer 1 25 ml 5 ml 2X Reaction Buffer 250 ul 1 ml DTT 1 M 125 ul 500 ul Caspase 3 Subsirate DEVD AFC 1 mM 5 ul 10 ul Caspase 3 Inhibitor DEVD CHO 1 mM 1 ml 4 ml AFC 80 uM Caspase 8 Fluorescent Assay Kits 25 assays 100 assays K2028 1 K2028 2 32 ml 125 ml Cell Lysis Buffer 1 25 ml 5 ml 2X Reaction Buffer 250 ul 1 ml DTT 1 M e 250 ul 1 ml Caspase 8 Substrate IETD AFC 1 mM 10 ul 20 ul Caspase 8 Inhibitor IETD fmk 1 mM e 1ml 4 ml AFC 80 uM Caspase 9 6 Fluorescent Assay Kits 25 assays 100 assays K2015 1 K2015 2 32 ml 125 ml Cell Lysis Buffer 1 5 ml 5ml 2X Reaction Buffer 250 ul 1 ml DTT 1 M 125 ul 500 ul Caspase 9 6 Substrate LEHD AMC 5 mM 20 ul 40 ul Caspase 9 6 Inhibitor LEHD CHO 5 mM 50 ul 150 ul DMSO lll Additional Materials Required The following materials are required but not supplied e Centrifuge for collecting cells Reaction tubes 0 5 1 5 ml or 96 well plates e DMSO for Caspase 3 and Caspase 8 assays AMC Sigma A9891 for Caspase 9 6 assay Fluorometer or 96 well plate reader Protocol PT3191 1 www bdbiosciences com BD Biosciences Clontech Version PR26395 5 BD ApoAlert Caspase Fluorescent Assay Kits User Manual IV BD ApoAlert Caspa
16. se 3 Assay Protocol PLEASE READ ENTIRE PROTOCOL BEFORE BEGINNING A General Considerations A relatively high concentration of DTT 10 mM is required for full activity of the enzymes Ensure that DTT is added to the Reaction Buffer when the assay is performed Otherwise unexpected low caspase activity will occur Turbidity or particulate materials in samples can decrease assay precision Reagents Aliquot a sufficient volume of 2X Reaction Buffer 50 ul assay for the number of assays you will perform Immediately before use add DTT to the aliquotted 2X Reaction Buffer add 10 ul of 1 M DTT stock per 1 ml of 2X Reaction Buffer Do not add DTT to stock 2X Reaction Buffer Turbidity or particulate materials in samples can decrease assay preci sion Ensure that all buffers are completely in solution You may need to heat free AFC to dissolve precipitates that can form during cold storage Controls We recommend performing three control reactions 1 a negative control on uninduced cells Step IV B 1 2 a control on induced cells treated with Caspase 3 Inhibitor Step IV B 6 3 a positive control for Caspase 3 induction You may treat your own cells with a known inducer At BD Biosciences Clontech we use Staurosporine 500 nM for 4 hr or human anti Fas monoclonal antibody clone CH 11 500 ng ml final concentration for 4 12 hr Anti Fas clone DX2 is not recommended for use with this kit Comparing the emission of an
17. trol reaction with purified Caspase 9 incubate an induced sample with Caspase 9 6 Inhibitor before adding substrate This reaction verifies that the signal detected by the kit is due to protease activity Add 50 ul of 2X Reaction Buffer DTT Mix Section VI A and 2 ul of Caspase 9 6 Inhibitor LEHD CHO to 50 ul of supernatant from a sample obtained in Step 5 Incubate on ice for 30 min together with the other samples Proceed to Step 7 where you will add reaction buffer to the remaining samples 7 Add 50 ul of 2X Reaction Buffer DTT Mix Section VI A to each reaction If you are using the Caspase 9 6 Inhibitor as a negative control add 2 ul DMSO per 50 ul of 2X Reaction Buffer to samples without inhibitor to ensure that all samples are tested under similar conditions 8 Add 5 ul of the 5 mM Caspase 9 6 Substrate LEHD AMC 250 uM final conc to each tube Incubate at 37 C for 1 hr or up to 3 hours maximum in a water bath Set up a parallel control reaction that does not contain conjugated substrate 9 Read in a fluorometer with a 380 nm excitation filter and 460 nm emission filter For plate reading transfer samples to a 96 well plate For quantification of protease activity proceed to Section VII EN Protocol PT3191 1 www bdbiosciences com BD Biosciences Clontech Version PR26395 BD ApoAlert Caspase Fluorescent Assay Kits User Manual Vil Quantification of protease activity for Caspase 3 amp 8 AFC Calibrat
18. ue necrosis factor TNF reviewed in Nicholson amp Thornberry 1997 Thornberry amp Littlewood 1998 Fraser amp Evan 1996 Cohen 1997 Upon activation by cell surface receptors such as Fas caspase 8 directly or indirectly initiates the proteolytic activities of downstream effector caspases such as caspase 3 Srinivasula et al 1996 Muzio et al 1997 Zou et al 1999 Caspase 9 another upstream caspase is activated via the mitochondrial release of cytochrome c to the cytosol Released cytochrome c binds to the apoptotic protease activating factor Apaf 1 in the cytosol forming a complex that activates procaspase 9 Zou et al 1999 Cain et al 1999 and Hu et al 1999 Active caspase 9 initiates a protease cascade that also activates caspase 3 Fernandes Alnemri et al 1994 and other downstream caspases Protocol PT3191 1 www bdbiosciences com BD Biosciences Clontech Version PR26395 3 BD ApoAlert Caspase Fluorescent Assay Kits User Manual l Introduction continued For a review of caspases and apoptosis see Green amp Reed 1998 Figure 1 illustrates how to detect protease activity using the BD ApoAlert Caspase Fluorescent Assay Kits The Caspase 3 and Caspase 8 Fluorescent Assay Kits detect the emission shift of 7 amino 4 trifluoromethyl coumarin AFC The AFC substrate conjugate usually emits blue light Amax 400 nm however cleavage of the substrate by the appropriate caspase liberates AFC which fluo
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