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AAT Bioquest/Amplite™ Fluorimetric Caspase 3/7 Assay Kit *Red Fluorescence*/13504/100 tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 534/619 |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | EnzymeDetection PeptidasesandProteases |
| Related | MicroplateReaders |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareCells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat20,000cells/well/90µLfora96-wellor5,000cells/well/20µLfora384-wellplateblackwall/clearbottomplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinculturemediumat80,000to200,000cells/well/90µLfora96-wellor20,000to50,000 cells/well/20µLfora384-wellblackwall/clearbottomplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforapoptosisinduction.
2.Preparestocksolutions:
2.1 ThawComponentA,B,C(ifdesired,ComponentD)atroomtemperaturebeforeuse.
2.2 Make200XZ-DEVD-ProRed™stocksolutionbyadding65µLDMSO(notprovided) intothevialofComponentA.
2.3 Ifdesired,preparea1mMstocksolutionofthecaspase3/7InhibitorAc-DEVD-CHO:Add100μLofDMSOdirectlytothevialofAc-DEVD-CHO(ComponentD).Thisinhibitorcanbeusedtoconfirmthecorrelationbetweenfluorescencesignalintensityandcaspase3/7-likeproteaseactivities.
Note:Theunusedinhibitorstocksolutionshouldbealiquotedandstoreddesiccatedat-20°C.
3.PrepareCaspase3/7assaysolution:
Add50µLof200XZ-DEVD-ProRed™stocksolution(fromStep2.2),and100µLof1MDTTsolution(ComponentC)into10mLAssayBuffer(ComponentB),andmixwell.
Note:50µLofthe200XZ-DEVD-ProRed™stocksolutionisenoughfor100assaysusingareactionvolumeof100μLperassay.Theunused200XZ-DEVD-ProRed™stocksolution(fromStep2.2)shouldbealiquoted,storeddesiccatedat-20°C,andprotectedfromlight.
4.RunCaspase3/7assay:
4.1 Treatcellsbyadding10µLof10Xtestcompounds(96-wellplate)or5µLof5Xtestcompounds(384-plate)intoPBSordesiredbuffer.Forblankwells(mediumwithoutthecells),addthesameamountofcompoundbuffer.
4.2 Incubatethecellplatesinanincubatorforadesiredperiodoftime(3-5hoursforJurkatcellstreatedwithstaurosporine)toinduceapoptosis.
4.3 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofcaspase3/7assaysolution(fromstep3).
4.4 Incubatetheplateatroomtemperatureforatleast1hour,keptfromlight.
Note1:Ifdesired,add1µLofthe1mMstocksolutionofthecaspase3/7InhibitorAc-DEVD-CHO(fromStep2.2)intoselectedsamples10minutesbeforeaddingthecaspase3/7assaysolutionatroomtemperaturetoconfirmthecaspase3/7-likeactivities.
4.5 MonitorthefluorescenceintensityatEx/Em=535/620nm(cutoffat610nm)witheithertoporbottomreadmode.
Note:Sometimes,bottomreadgivesbettersignaltobackgroundratio,centrifugecellplate(especiallyforthenon-adherentcells)at800rpmfor2minutes(brakeoff)ifusingbottomreadmode.
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