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AAT Bioquest/Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Green Fluorescence*/22799/200 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.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°Cincubatorforadesiredperiodoftimetoinduceapoptosis.
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=490/525nm.
| References&Citations |  PrinterFriendlyVersion |
1. KemnitzerW,KuemmerleJ,JiangS,SirisomaN,KasibhatlaS,Crogan-GrundyC,TsengB,DreweJ,CaiSX.(2009)Discoveryof1-benzoyl-3-cyanopyrrolo[1,2-a]quinolinesasanewseriesofapoptosisinducersusingacell-andcaspase-basedhigh-throughputscreeningassay.2:Structure-activityrelationshipsofthe4-,5-,6-,7-and8-positions.BioorgMedChemLett,19,3481.
2. KemnitzerW,SirisomaN,NguyenB,JiangS,KasibhatlaS,Crogan-GrundyC,TsengB,DreweJ,CaiSX.(2009)DiscoveryofN-aryl-9-oxo-9H-fluorene-1-carboxamidesasanewseriesofapoptosisinducersusingacell-andcaspase-basedhigh-throughputscreeningassay.1.Structure-activityrelationshipsofthecarboxamidegroup.BioorgMedChemLett,19,3045.
3. SchmidtMF,El-DahshanA,KellerS,RademannJ.(2009)Selectiveidentificationofcooperativelybindingfragmentsinahigh-throughputligationassayenablesdevelopmentofapicomolarcaspase-3inhibitor.AngewChemIntEdEngl,48,6346.
4. SirisomaN,PervinA,DreweJ,TsengB,CaiSX.(2009)DiscoveryofsubstitutedN"-(2-oxoindolin-3-ylidene)benzohydrazidesasnewapoptosisinducersusingacell-andcaspase-basedHTSassay.BioorgMedChemLett,19,2710.
5. SirisomaN,PervinA,NguyenB,Crogan-GrundyC,KasibhatlaS,TsengB,DreweJ,CaiSX.(2009)Discoveryofsubstituted4-anilino-2-arylpyrimidinesasanewseriesofapoptosisinducersusingacell-andcaspase-basedhighthroughputscreeningassay.2.Structure-activityrelationshipsofthe2-arylgroup.BioorgMedChemLett,19,2305.
6. CursioR,ColosettiP,AubergerP,GugenheimJ.(2008)Liverapoptosisfollowingnormothermicischemia-reperfusion:invivoevaluationofcaspaseactivitybyFLIVOassayinrats.TransplantProc,40,2038.
7. JiangS,Crogan-GrundyC,DreweJ,TsengB,CaiSX.(2008)Discoveryof(naphthalen-4-yl)(phenyl)methanonesandN-methyl-N-phenylnaphthalen-1-aminesasnewapoptosisinducersusingacell-andcaspase-basedHTSassay.BioorgMedChemLett,18,5725.
8. KemnitzerW,DreweJ,JiangS,ZhangH,Crogan-GrundyC,LabrequeD,BubenickM,AttardoG,DenisR,LamotheS,GourdeauH,TsengB,KasibhatlaS,CaiSX.(2008)Discoveryof4-aryl-4H-chromenesasanewseriesofapoptosisinducersusingacell-andcaspase-basedhighthroughputscreeningassay.4.Structure-activityrelationshipsofN-alkylsubstitutedpyrrolefusedatthe7,8-positions.JMedChem,51,417.
9. KemnitzerW,JiangS,WangY,KasibhatlaS,Crogan-GrundyC,BubenikM,LabrecqueD,DenisR,LamotheS,AttardoG,GourdeauH,TsengB,DreweJ,CaiSX.(2008)Discoveryof4-aryl-4H-chromenesasanewseriesofapoptosisinducersusingacell-andcaspase-basedHTSassay.Part5:modificationsofthe2-and3-positions.BioorgMedChemLett,18,603.
10. KemnitzerW,JiangS,ZhangH,KasibhatlaS,Crogan-GrundyC,BlaisC,AttardoG,DenisR,LamotheS,GourdeauH,TsengB,DreweJ,CaiSX.(2008)Discoveryof4-aryl-2-oxo-2H-chromenesasanewseriesofapoptosisinducersusingacell-andcaspase-basedhigh-throughputscreeningassay.BioorgMedChemLett,18,5571.
