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AAT Bioquest/Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Blue Fluorescence Excited at 405 nm*/22835/100 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 405/450 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellCytotoxicity |
| Related | ApoptosisandCytotoxicity CellApoptosis BiochemicalAssays |
1.PrepareandincubatecellswithApopxin™Violet450:
1.1 Treatcellswithtestcompoundsforadesiredperiodoftime(4-6hoursforJurkatcellstreatedwithstaurosporine)toinduceapoptosis.
1.2 Centrifugethecellstoget1-5×105cells/tube.
1.3 ResUSPendcellsin200μLofAssayBuffer(ComponentB).
1.4 Add2μLofApopxin™Violet450(ComponentA)intothecells.
Optional:Add2µLof100XPropidiumIodide(ComponentC)intothecellsfornecrosiscells.
1.5 Incubateatroomtemperaturefor30to60minutes,protectedfromlight.
1.6 Add300μLofAssayBuffer(ComponentB)toincreasevolumebeforeanalyzingthecellswithaflowcytometerorfluorescencemicroscope(seeStep1.7below).
1.7 MonitorthefluorescenceintensityatEx/Em=405/450nmbyusingaflowcytometerorafluorescencemicroscope(SeeStep2or3below).
2.Analyzebyusingaflowcytometer:
QuantifyApopxin™Violet450bindingbyusingaflowcytometeratEx/Em=405/450nm.MeasurethecellviabilitybyusingtheFL2channelwhenpropidiumiodideisaddedintothecells.
Note:Apopxin™bindingflowcytometricanalysisonadherentcellsisnotroutinelytestedsincespecificmembranedamagemayoccurduringcelldetachmentorharvesting.However,methodsforutilizingAnnexinVforflowcytometryonadherentcelltypeshavebeenpreviouslyreportedbyCasiola-Rosenetal.andvanEngelendetal(seeRefs1and2).
3.Analyzebyusingafluorescencemicroscope:
3.1 PipettethecellsuspensionfromStep1.5,rinse1-2timeswithassaybuffer,andthenresuspendthecellswithassaybuffer.Addthecellsonaglassslidethatiscoveredwithaglasscoverslip.
Note:Foradherentcells,itisrecommendedtogrowthecellsdirectlyonacoverslip.AfterincubationwithApopxin™Violet450(Step1.5),rinse1-2timeswithassaybuffer,andaddassaybufferbacktothecoverslip.Invertcoversliponaglassslideandvisualizethecells.Thecellscanalsobefixedin2%formaldehydeaftertheincubationwithApopxin™Violet450andvisualizedunderamicroscope.
3.2 AnalyzetheapoptoticcellswithApopxin™Violet450underafluorescencemicroscopeusingtheVioletchannel.MeasurethecellviabilitybyusingtheTRITCchannelwhenpropidiumiodideisaddedintothecells.ThebluestainingontheplasmamembraneindicatestheApopxin™Violet450bindingtoPSoncellsurface.
| References&Citations |  PrinterFriendlyVersion |
1. BaeON,LimKM,NohJY,ChungSM,KimSH,ChungJH.(2009)Trivalentmethylatedarsenical-inducedphosphatidylserineexposureandapoptosisinplateletsmayleadtoincreasedthrombusformation.ToxicolApplPharmacol,239,144.
2. BurteaC,LaurentS,LancelotE,BalletS,MurariuO,RousseauxO,PortM,VanderElstL,CorotC,MullerRN.(2009)PeptidictargetingofphosphatidylserinefortheMRIdetectionofapoptosisinatheroscleroticplaques.MolPharm,6,1903.
3. DongHP,HolthA,KleinbergL,RuudMG,ElstrandMB,TropeCG,DavidsonB,RisbergB.(2009)Evaluationofcellsurfaceexpressionofphosphatidylserineinovariancarcinomaeffusionsusingtheannexin-V/7-AADassay:clinicalrelevanceandcomparisonwithotherapoptosisparameters.AmJClinPathol,132,756.
4. LiuT,ZhuW,YangX,ChenL,YangR,HuaZ,LiG.(2009)DetectionofapoptosisbasedontheinteractionbetweenannexinVandphosphatidylserine.AnalChem,81,2410.
5. MirnikjooB,BalasubramanianK,SchroitAJ.(2009)Suicidalmembranerepairregulatesphosphatidylserineexternalizationduringapoptosis.JBiolChem,284,22512.
6. MirnikjooB,BalasubramanianK,SchroitAJ.(2009)Mobilizationoflysosomalcalciumregulatestheexternalizationofphosphatidylserineduringapoptosis.JBiolChem,284,6918.
7. ThapaN,KimS,SoIS,LeeBH,KwonIC,ChoiK,KimIS.(2008)Discoveryofaphosphatidylserine-recognizingpeptideanditsutilityinmolecularimagingoftumourapoptosis.JCellMolMed,12,1649.
8. AiresV,HichamiA,FilomenkoR,PleA,RebeC,BettaiebA,KhanNA.(2007)Docosahexaenoicacidinducesincreasesin[Ca2+]iviainositol1,4,5-triphosphateproductionandactivatesproteinkinaseCgammaand-deltaviaphosphatidylserinebindingsite:implicationinapoptosisinU937cells.MolPharmacol,72,1545.
9. BalasubramanianK,MirnikjooB,SchroitAJ.(2007)Regulatedexternalizationofphosphatidylserineatthecellsurface:implicationsforapoptosis.JBiolChem,282,18357.
10. CauchonN,LangloisR,RousseauJA,TessierG,CadoretteJ,LecomteR,HuntingDJ,PavanRA,ZeislerSK,vanLierJE.(2007)PETimagingofapoptosiswith(64)Cu-labeledstreptavidinfollowingpretargetingofphosphatidylserinewithbiotinylatedannexin-V.EurJNuclMedMolImaging,34,247.
