| 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.PrepareandincubatecellswithApopxin™DeepRed:
1.1 Treatcellswithtestcompoundsforadesiredperiodoftime(4-6hoursforJurkatcellstreatedwithstaurosporine)toinduceapoptosis.
1.2 Centrifugethecellstoget1-5×105cells/tube.
1.3 ResUSPendcellsin200μLofAssayBuffer(ComponentB).
1.4 Add2μLofApopxin™Red(ComponentA)intothecells.
Optional1:Add1µLof200XNuclearGreen™DCS1(ComponentC)intothecellsfornecrosiscells.
Optional2:Add100µLofDMSOintothevialofCytoCalcein™Violet450(ComponentD)tohave200XCytoCalcein™Violet450stocksolution,andthenadd1µLintothecellsforhealthycellsstaining.
1.5 Incubateatroomtemperaturefor30to60minutes(protectedfromlight).
1.6 Add300μLofAssayBuffer(ComponentB)toincreasevolumebeforeanalyzingthecellswithaflowcytometerorfluorescencemicroscope(seeStep1.7below).
1.7 MonitorthefluorescenceintensityatEx/Em=630/660nmforapoptosis,490/520nmfornecrosis,and405/450nmforhealthycellsusingaflowcytometerorafluorescencemicroscope(SeeStep2or3below).
2.Analyzecellsusingaflowcytometer:
QuantifyApopxin™RedbindingbyusingtheFL4channel(Ex/Em=630/660nm),andmeasurethecellviabilityusingtheFL1channel(Ex/Em=490/520nm)whenNuclearGreen™DCS1isadded,and/orusingEx/Em=405/450nmwhenCytoCalcein™Violet450isaddedintothecells.
Note:TheflowcytometricanalysisofApopxin™bindingtoadherentcellsisnotroutinelytestedsincespecificmembranedamagemayoccurduringcelldetachmentorharvesting.However,methodsforutilizingAnnexinVforflowcytometryonadherentcelltypeshavebeenpreviouslyreportedbyCasiola-Rosenetal.andvanEngelendetal(seeRefs1and2).
3.Analyzecellsusingafluorescencemicroscope:
3.1 PipettethecellsuspensionfromStep1.5,rinse1-2timeswithassaybuffer,andthenresuspendthecellswithassaybuffer.Addthecellsonaglassslidethatiscoveredwithaglasscoversliporablackwall/clearbottom96-wellmicroplate.
Note:Foradherentcells,itisrecommendedtogrowthecellsdirectlyonacoverslip(orablackwall/clearbottom96-wellmicroplate).AfterincubationwithApopxin™DeepRed(Step1.5),rinse1-2timeswithassaybuffer,andthenaddassaybufferbacktothecoverslip(orablackwall/clearbottom96-wellmicroplate).Invertcoversliponaglassslideandvisualizethecells.Thecellscanalsobefixedin2%formaldehydeaftertheincubationwithApopxin™DeepRedandvisualizedunderamicroscope.
3.2 AnalyzetheapoptoticcellswithApopxin™DeepRedunderafluorescencemicroscopeusingtheCy5channel.MeasurethecellviabilityusingtheFITCchannelwhenNuclearGreen™DCS1isadded,and/orVioletchannelwhenCytoCalcein™Violet450isaddedintothecells.TheredstainingontheplasmamembraneindicatestheApopxin™DeepRedbindingtoPSoncellsurface.
| References&Citations |  CitationExplorer |
Anthocyanin-richblackcurrantextractinhibitsproliferationoftheMCF10AhealthyhumanbreastepithelialcelllinethroughinductionofG0/G1arrestandapoptosis
Authors:NaokiNanashima,KayoHorie,MitsuruChiba,ManabuNakano,HayatoMaeda,ToshiyaNakamura
Journal:MolecularMedicineReports(2017):6134--6141
ClusterinsignalsviaApoER2/VLDLRandinducesmeiosisofmalegermcells
Authors:MuhammadAssadRiaz,AngelikaStammler,MareikeBorgers,LutzKonrad
Journal:AmericanJournalofTranslationalResearch(2017):1266
DetectingApoptosis,Autophagy,andNecrosis
Authors:JackColeman,RuiLiu,KathyWang,ArunKumar
Journal:ApoptosisMethodsinToxicology(2016):77--92
