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AAT Bioquest/Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Blue Fluorescence*/22813/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 351/430 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellCytotoxicity |
| Related | ApoptosisandCytotoxicity CellApoptosis BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparecells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat20,000cells/well/90µLfora96-wellplateor5,000cells/well/20µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinculturemediumat200,000cells/well/90µLfora96-wellpoly-Dlysineplateor50,000cells/well/20µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforapoptosisinduction.
2.Preparecaspase9assayloadingsolution:
2.1 Thawallthekitcomponentstoroomtemperaturebeforeuse.
2.2 Makecaspase9assayloadingsolution:Add50µLofCaspase9Substrate(ComponentA)into10mLofAssayBuffer(ComponentB),andmixthemwell.
Note:AliquotandstoretheunusedCaspase9Substrate(ComponentA)andAssayBuffer(ComponentB)at-20oC.Avoidrepeatedfreeze/thawcycles.
3B3.Runcaspase9assay:
3.1 Treatcellsbyadding10µL/wellof10Xtestcompounds(96-wellplate)or5µL/wellof5Xtestcompounds(384-wellplate)intoPBSorthedesiredbuffer.Forblankwells(mediumwithoutthecells),addthesameamountofcompoundbuffer.
3.2 Incubatethecellplateina5%CO2,37°Cincubatorforadesiredperiodoftime(4-6hoursforJurkatcellstreatedwithstaurosporine)toinduceapoptosis.
3.3 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofcaspase9assayloadingsolution(fromStep2.2).
3.4 Incubatetheassayloadingsolutionplateatroomtemperatureforatleast1hour,protectedfromlight.
Note:Ifdesired,add1µLofthe1mMAc-LEHD-CHOcaspase9inhibitortoselectedsamples10minutesbeforeaddingtheassayloadingsolutionatroomtemperaturetoconfirmtheinhibitionofthecaspase9-likeactivity.
3.5 Centrifugethecellplate(especiallyforthenon-adherentcells)at800rpmfor2minutes(brakeoff).
3.6 MonitorthefluorescenceintensityatEx/Em=375/435nm(cutoff420nm)
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