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AAT Bioquest/Amplite™ Fluorimetric Caspase 3/7 Assay Kit *Blue Fluorescence*/13502/500 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 351/430 |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | EnzymeDetection PeptidasesandProteases |
| Related | MicroplateReaders |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareCells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat20,000to80,000cells/well/90µLfora96-wellplateor5,000to20,000cells/well/20µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinculturemediumat80,000to200,000cells/well/90µLfora96-wellpoly-Dlysineplateor20,000to50,000cells/well/20µLfora384-wellpoly-Dlysineplate.Centrifugetheplatesat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforapoptosisinduction.
2.Preparestocksolutions:
2.1 ThawComponentA,B,C(ifdesired,ComponentD)atroomtemperaturebeforeuse.
2.2 Ifdesired,preparea1mMstocksolutionoftheCaspase3/7InhibitorAc-DEVD-CHO:Add100μLofDMSO(notprovided)directlytothevialofCaspase3/7InhibitorAc-DEVD-CHO(ComponentD).ThisinhibitorcanbeusedtoconfirmthecorrelationbetweenfluorescencesignalintensityandCaspase3/7-likeproteaseactivities.
Note:Theun-usedinhibitorstocksolutionshouldbealiquotedandstoreddesiccatedat-20°C.
3.PrepareCaspase3/7assaysolution:
Add50µLof200XCaspase3/7Substratestocksolution(ComponentA),and100µLof1MDTTsolution(ComponentC)into10mLofAssaybuffer(ComponentB),andmixwell.
Note:50µLofthe200XCaspase3/7Substratestocksolutionisenoughfor100assaysusingareactionvolumeof100μLperassay.Theunused200XCaspase3/7Substratestocksolutionshouldbealiquotedandstoreddesiccatedat-20°C.Keepfromlight.
4.RunCaspase3/7assay:
4.1 Treatcellsbyadding10µLof10Xtestcompounds(96-wellplate)or5µLof5Xtestcompounds(384-plate)inPBSordesiredbuffer.Forblankwells(mediumwithoutthecells),addthecorrespondingamountofcompoundbuffer.
4.2 Incubatethecellplateina37°C,5%CO2incubatorforadesiredperiodoftime(4-6hoursforJurkatcellstreatedwithcamptothecin)toinduceapoptosis.
4.3 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofCaspase3/7assaysolution(fromStep3).
4.4 Incubatetheplateatroomtemperatureforatleast1hour,protectedfromlight.
Note1:Ifdesired,add1µLofthe1mMstocksolutionoftheCaspase3/7InhibitorAc-DEVD-CHO(fromStep2.2)toselectedsamples10minutesbeforeaddingtheassaysolutionatroomtemperaturetoconfirmthecaspase3/7-likeactivities.
4.5 Centrifugethecellplate(especiallyforthenon-adherentcells)at800rpmfor2minuteswithbrakeoff.
4.6 MonitorthefluorescenceincreaseatEx/Em=350/450nm.
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