| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 424/560 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellCytotoxicity |
| Related | ApoptosisandCytotoxicity CellApoptosis BiochemicalAssays |
1.PrepareandincubatecellswithAnnexinV-mFluorViolet™550:
1.1 Treatcellswithtestcompoundsforadesiredperiodoftime(4-6hoursforJurkatcellstreatedwithstaurosporine)toinduceapoptosis.
1.2 Centrifugethecellstoget1-5×105cells/tube.
1.3 ResUSPendcellsin200μLofAssayBuffer(ComponentB).
1.4 Add2μLofAnnexinV-mFluorViolet™550(ComponentA)intothecells.
Optional:Add2µLof100XPropidiumIodide(ComponentC)intothecellsfornecrosiscells.
1.5 Incubateatroomtemperaturefor30to60minutes,protectedfromlight.
1.6 Add300μLofAssayBuffer(ComponentB)toincreasevolumebeforeanalyzingthecellswithaflowcytometerorfluorescencemicroscope(seeStep1.7below).
1.7 MonitorthefluorescenceintensityatEx/Em=405/550nmbyusingaflowcytometerorafluorescencemicroscope(SeeStep2or3below).
2.Analyzebyusingaflowcytometer:
QuantifyAnnexinV-mFluorViolet™550bindingbyusingaflowcytometeratEx/Em=405/550nm.MeasurethecellviabilitybyusingtheFL2channelwhenpropidiumiodideisaddedintothecells.
Note:AnnexinVbindingflowcytometricanalysisonadherentcellsisnotroutinelytestedsincespecificmembranedamagemayoccurduringcelldetachmentorharvesting.However,methodsforutilizingAnnexinVforflowcytometryonadherentcelltypeshavebeenpreviouslyreportedbyCasiola-Rosenetal.andvanEngelendetal(seeRefs1and2).
3.Analyzebyusingafluorescencemicroscope:
3.1 PipettethecellsuspensionfromStep1.5,rinse1-2timeswithassaybuffer,andthenresuspendthecellswithassaybuffer.Addthecellsonaglassslidethatiscoveredwithaglasscoverslip.
Note:Foradherentcells,itisrecommendedtogrowthecellsdirectlyonacoverslip.AfterincubationwithAnnexinV-mFluorViolet™550(Step1.5),rinse1-2timeswithassaybuffer,andaddassaybufferbacktothecoverslip.Invertcoversliponaglassslideandvisualizethecells.Thecellscanalsobefixedin2%formaldehydeaftertheincubationwithAnnexinV-mFluorViolet™550andvisualizedunderamicroscope.
3.2 AnalyzetheapoptoticcellswithAnnexinV-mFluorViolet™550underafluorescencemicroscopeusingtheVioletchannel.MeasurethecellviabilitybyusingtheTRITCchannelwhenpropidiumiodideisaddedintothecells.TheorangestainingontheplasmamembraneindicatestheAnnexinV-mFluorViolet™550bindingtoPSoncellsurface.
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