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AAT Bioquest/Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Red Fluorescence*/22816/100 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 534/619 |
| 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-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.Preparecaspase8assayloadingsolution:
2.1 Thawbothofthekitcomponentsatroomtemperaturebeforeuse.
2.2 Make200XAc-IETD-ProRed™stocksolutionbyadding65µLDMSOintothevialofComponentA.
2.3 Makecaspase8assayloadingsolutionbyadding50µLofAc-IETD-ProRed™Stocksolution(200X,fromStep
2.2) into10mLofAssayBuffer(ComponentB),andmixthemwell.
Note1:Caspasae8assayloadingsolutionisnotstable,shouldbeusedpromptly.
Note2:Aliquotandstoreunusedcaspase8substrate(ComponentA)andassaybuffer(ComponentB)at
-20oC.Avoidrepeatedfreeze/thawcycles.
3B3.Assayprocedures:
3.1 Treatcellsbyadding10µL/wellof10Xtestcompounds(96-wellplate)or5µL/wellof5Xtestcompounds(384-plate)intoPBSorthedesiredbuffer.Forblankwells(mediumwithoutthecells),addthesameamountofcompoundbuffer.
3.2 Incubatethecellplateina37°C,5%CO2,incubatorforadesiredperiodoftime(3-4hoursforJurkatcellstreatedwithstaurosporine)toinduceapoptosis.
3.3 Add100µL/well/96-welor25µL/well/384-wellplateofcaspase8assayloadingsolution(fromStep2.3).
3.4 Incubatetheplateatroomtemperatureforatleast1hour,protectedfromlight.
Note:Ifdesired,add1µLofthe1mMAc-IETD-CHOcaspase8inhibitortoselectedsamples10minutesbeforeaddingtheassayloadingsolutionatroomtemperaturetoconfirmtheinhibitionofthecaspase8-likeactivities.
3.5 MonitorthefluorescenceintensityatEx/Em=535/620nm(cutoffat610nm)witheithertoporbottomreadmode.
Note:Sometimes,bottomreadgivesbettersignaltobackgroundratio,centrifugecellplate(especiallyforthenon-adherentcells)at800rpmfor2minutes(brakeoff)ifusingbottomreadmode.
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