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AAT Bioquest/Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Green Fluorescence*/22798/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 498/520 |
| 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.Preparecaspase8assayloadingsolution:
2.1 Thawbothofthekitcomponentstoroomtemperaturebeforeuse.
2.2 Preparecaspase8assayloadingsolution:Add50µLofCaspase8Substrate(ComponentA)into10mLofAssayBuffer(ComponentB),andmixthemwell.
Note1:Caspasae8assayloadingsolutionisnotstable,usepromptly.
Note2:AliquotandstoreunusedCaspase8Substrate(ComponentA)andAssayBuffer(ComponentB)at
-20oC.Avoidrepeatedfreeze/thawcycles.
3.Runcaspase8assay:
3.1 Treatcellsbyadding10µL/wellof10Xtestcompounds(96-wellplate)or5µL/wellof5Xtestcompounds(384-wellplate)intoPBSorthedesiredbuffer.Forblankwells(mediumwithoutthecells),addthesameamountofcompoundbuffer.
3.2 Incubatethecellplateina5%CO2,37°Cincubatorforadesiredperiodoftime(4-6hoursforJurkatcellstreatedwithcamptothecinorstaurosporine)toinduceapoptosis.
3.3 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofcaspase8assayloadingsolution(fromStep2.2).
3.4 Incubatethecaspase8assayloadingsolutionplateatroomtemperaturefor30minto1hour,protectedfromlight.
Note:Ifdesired,add1µLofthe1mMAc-IETD-CHOcaspase8inhibitortoselectedsamples10minutesbeforeaddingtheassayloadingsolutionatroomtemperaturetoconfirmtheinhibitionofthecaspase8-likeactivities.
3.5 Centrifugecellplate(especiallyforthenon-adherentcells)at800rpmfor2minutes(brakeoff).
3.6 MonitorthefluorescenceincreaseatEx/Em=490/525nm.
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