| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 638/655 |
| MW | N/A |
| CAS# | N/A |
| Solvent | Water |
| Storage | F/D/L |
| Category | CellAnalysis CellApoptosis |
| Related | ApoptosisandCytotoxicity BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareandincubatecellswithAnnexinVconjugates:
1.1 PrepareAnnexinV-bindingassaybuffer:10mMHEPES,140mMNaCl,and2.5mMCaCl2,pH7.4.
1.2 Treatcellswithtestcompoundsforadesiredperiodoftime(4-6hoursforJurkatcellstreatedwithstaurosporine)toinduceapoptosis.
1.3 Centrifugethecellstoget1-5×105cells/tube.
1.4 ResUSPendcellsin200μLofAnnexinV-bindingassaybuffer(fromStep1.1).
1.5 Add2μLofAnnexinVconjugateintothecells.
Optional:AddadeadcellstainsuchasPropidiumIodideintothecellsfornecrosiscells.
1.6 Incubateatroomtemperaturefor30to60minutes,protectedfromlight.
1.7 Add300μLofAnnexinV-bindingassaybuffer(fromStep1.1)toincreasevolumebeforeanalyzingthecellswithaflowcytometerorfluorescencemicroscope(seeStep1.8below).
1.8 Monitorthefluorescenceintensitybyusingaflowcytometerorafluorescencemicroscope(SeeStep2or3below).
2.Analyzebyusingaflowcytometer:
QuantifyAnnexinVconjugatesbindingbyusingaflowcytometerwithappropriatedfilters.
Note:AnnexinVbindingflowcytometricanalysisonadherentcellsisnotroutinelytestedsincespecificmembranedamagemayoccurduringcelldetachmentorharvesting.However,methodsforutilizingAnnexinVforflowcytometryonadherentcelltypeshavebeenpreviouslyreportedbyCasiola-Rosenetal.andvanEngelendetal(seeRefs1and2).
3.Analyzebyusingafluorescencemicroscope:
3.1 PipettethecellsuspensionfromStep1.6,rinse1-2timeswithAnnexinV-bindingassaybuffer(fromStep1.1),andthenresuspendthecellswiththeAnnexinV-bindingassaybuffer(fromStep1.1).Addthecellsonaglassslidethatiscoveredwithaglasscoverslip.
Note:Foradherentcells,itisrecommendedtogrowthecellsdirectlyonacoverslip.AfterincubationwithAnnexinVconjugate(Step1.6),rinse1-2timeswithAnnexinV-bindingassaybuffer(fromStep1.1),andaddAnnexinV-bindingassaybuffer(fromStep1.1)backtothecoverslip.Invertcoversliponaglassslideandvisualizethecells.Thecellscanalsobefixedin2%formaldehydeaftertheincubationwithAnnexinVconjugateandvisualizedunderamicroscope.
3.2 AnalyzetheapoptoticcellswithAnnexinVconjugateunderafluorescencemicroscopewithappropriatedfilters.
| References&Citations |  CitationExplorer |
Resources-ApplicationNotesPhenotypicandEpigeneticMechanismofActionDeterminationsofHistoneMethylaseandDemethylaseInhibitorsusingDigitalWidefieldMicroscopy
Authors:BradLarson,PeterBanks
Journal:ExperimentalBIOLOGy(2017)
TargetedMagneticIntra-LysosomalHyperthermiaproduceslysosomalreactiveoxygenspeciesandcausesCaspase-1dependentcelldeath
Authors:PascalClerc,PaulineJeanjean,NicolasHalalli,MichelGougeon,BernardPipy,JulianCarrey,DanielFourmy,VéroniqueGigoux
Journal:JournalofControlledRelease(2017)
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