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AAT Bioquest/Cell Meter™ Multiplexing Caspase 3/7, 8 and 9 Activity Assay Kit *Triple Fluorescence Colors*/22820/100 T
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellCytotoxicity |
| Related | ApoptosisandCytotoxicity CellApoptosis BiochemicalAssays |
1.Preparecells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat20,000cells/well/90µLfora96-wellor5,000cells/well/20µLfora384-wellplateblackwall/clearbottomplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinculturemediumat200,000cells/well/90µLfora96-wellor50,000cells/well/20µLfora384-wellblackwall/clearbottomplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforapoptosisinduction.
1.3 Treatcellsbyadding10µL/wellof10Xtestcompounds(96-wellplate)or5µL/wellof5Xtestcompounds(384-plate)intoPBSorthedesiredbuffer.Forblankwells(mediumwithoutthecells),addthesameamountofcompoundbuffer.
1.4 Incubatethecellplateina37°C,5%CO2,incubatorforadesiredperiodoftime(3-5hoursforJurkatcellstreatedwithstaurosporine)toinduceapoptosis.
2. Preparecaspaseassayloadingsolution:
2.1 Thawkitcomponentsatroomtemperaturebeforeuse.
2.2 Toassaysinglecaspaseactivityineachwell:Makecaspase3/7,caspase8orcaspase9assayloadingsolutionbyadding50µLofsubstrate(ComponentA,BorC)into10mLofAssayBuffer(ComponentD),andmixthemwell.
2.3 Toassaydual-ortri-caspaseactivityinthesamewell: Add50µLofeachinterestedcaspasesubstrateinto10mLofAssayBuffer(ComponentD)togethertomaketheassayloadingsolution.
Note:Pleasepreparethetestedsubstratesolutionsandtheneededvolumeproportionally,storetheunusedsubstratestocksolutionat-20oC. Avoidrepeatedfreeze/thawcycles.
3. RunAssay:
3.1 Add100mL/well/96-welor25µL/well/384-wellplateofcaspaseassayloadingsolution(fromstep2.3).
3.2 Incubatetheplateatroomtemperatureforatleast30to60min,protectedfromlight.
Note:Ifdesired,add1µLof1mMcaspaseinhibitortoselectedsamples10minutesbeforeaddingtheassayloadingsolutionatroomtemperaturetoconfirmtheinhibitionofcaspaseactivities.
3.3 Monitorthefluorescenceintensityasindicatedinthetablewitheithertoporbottomreadmode.
Caspasetobeassayed | Ex/Em |
Caspase3/7, Redfluorescence | 535/620nm |
Caspase8,Greenfluorescence | 490/525nm |
Caspase9,Bluefluorescence | 370/450nm |
Note:Sometimes,bottomreadgivesbettersignaltobackgroundratio,centrifugecellplate(especiallyforthenon-adherentcells)at800rpmfor2minutes(brakeoff)ifusingbottomreadmode.
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