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AAT Bioquest/Cell Meter™ No-Wash Live Cell Caspase 3/7 Activity Assay Kit *Red Fluorescence*/20260/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 405/600 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellApoptosis |
| Related | ApoptosisandCytotoxicity |
1. Culturecellstoadensityoptimalforapoptosisinductionaccordingtoyourspecificinductionprotocol,butnottoexceed2x106cells/mLforsUSPensioncells.Foradherentcells,platecellsat30,000-80,000cells/well/96-wellplateovernight.
2. Make1XApoBrite™V570Caspase3/7substrateworkingsolutionbyadding2µLof500XApoBrite™caspasesuV570Caspase3/7substrateDMSOstocksolution(ComponentA)into1mLHHBS(ComponentB).
Note:Theunused500XApoBrite™V570Caspase3/7substrateDMSOstocksolutionshouldbedividedassingleusealiquotandstoredat-20°C.
3. Removegrowthmedium,andwashoncewithHHBS(ComponentB)orbufferofyourchoice.
4. Add100µL/well/96wellplateof1XApoBrite™V570Caspase3/7substrateworkingsolution(fromStep2),andincubateat37ºC,5%CO2incubatorfor2hours.
5. RemoveApoBrite™V570Caspase3/7substrateworkingsolutionandwashoncewithHHBS(ComponentB).
6. AddgrowthmediumbacktotheApoBrite™V570caspase3/7loadedcells(fromStep5),andtreatthecellsasdesiredforapoptosis.Hereareafewexamplesfor inducingapoptosisincellculture:
1)TreatingJurkatcellswith2μg/mlcamptothecinfor3hours.
2)TreatingJurkatcellswith1μMstaurosporinefor3hours.Helacellsfor1hour.
3)TreatingHL-60cellswith4μg/mlcamptothecinfor4hours.
4)TreatingHL-60cellswith1μMstaurosporinefor4hours.
5)TreatingHelacellswith1μMstaurosporinefor1hour.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforapoptosisinduction.
7. Ifdesired,labelthecellswithaDNAstain(suchas7-AADfordeadcells)
8. Monitorthefluorescenceintensitybyfluorescencemicroscopy,flowcytometer,orfluorescentmicroplatereaderatEx/Em=405/570nm(for7-AAD,Ex/Em=535/650nm)
8.1 Forflowcytometry(suspensioncells),monitorthefluorescenceintensityusingEx/Em=405/570nmfilters(FL3channelfor7-AADstaining).Gateonthecellsofinterest,excludingdebris.
8.2 Forfluorescencemicroscopy,observecellsunderafluorescencemicroscopeusingEx/Em=405/570nm orDAPIchannel(TRITCchannelfor7-AADstaining).
8.3 Forfluorescentmicroplatereader,monitorthefluorescenceintensityusingEx/Em=405/570nm(cutoffat540nm)bottomreadmode.
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