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A general protocol for staining cell for cytometry analysis
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GeneralAnnexinVStainingProcedure Solutions 1. 10XBindingBuffer(Cat.No.66121A):0.1MHEPES,pH7.4;1.4MNaCl;25mMCaCl2.Diluteto1Xpriortouse. 2. PropidiumIodide(PI).Preparea50µg/mlstocksolutionofPIin1XPBSbuffer.StoreatRT.RecommendedforuseinparallelwithAnnexinV-FITC(Cat.No.65874X,65874H)orAnnexinV-Biotin(Cat.Nos.65872X,65872H). 3. Via-Probe™(Cat.No.34321X):Aconvenientready-to-usesolutionofthenucleicaciddye7-AAD.RecommendedforuseinparallelwithAnnexinV-PE(Cat.No.65875X,65875H)orAnnexinV-Biotin(Cat.Nos.65872X,65872H). 4. 1XPBSBuffer:8gNaCl;0.2gKCl;1.44gNa2HPO4•7H20;0.24gKH2PO4;H20to1liter.AdjustpHto7.2,autoclaveandstoreatRT. Staining 1. WashcellstwicewithcoldPBSandthenresUSPendcellsin1XBindingBufferataconcentrationof~1x106cells/ml. 2. Transfer100µlofthesolution(~1x105cells)toa5mlculturetube. 3. AddAnnexinVandVitalDyeasdescribedbelow: *TheoptimalamountofPImayrangebetween2–10µl/testdependingoncelltypeandexperimentalsystem.2µl/testistherecommendedstartingamount. 4. Gentlymixthecellsandincubatefor15minatRTinthedark. *ForAnnexinV-Biotinsamplesonly:After15minincubation,washoncewith1mlof1XBindingBuffer.Dilute0.5µgStreptavidin-FluoresceinIsothiocynate(SAv-FITC,Cat.No.13024D)into100µlof1XBindingBufferandaddtothecellpellet.Gentlymixthecells.Add2µlPIandincubatefor15minatRT. 5. Add400µlof1XBindingBuffertoeachtube.AnalyzebyflowcytometryassoonaspossIBLe(within1hr). Note:MethodsforutilizingAnnexinVbindingonadherentcells(i.e.,monolayer)havebeendescribedbyvanEngelandetal.16andCasciola-Rosenetal.1However,thesemethodsarenotperformedasaroutinequalitycontrolfortheAnnexinV-FITCApoptosisDetectionKitIandKitII. SuggestedControlsforFlowCytometricAnalysisofAnnexinVsamples. Thefollowingcontrolsareusedtosetupcompensationandquadrants: FITC 1. Unstainedcells 2. CellsstainedwithAnnexinV-FITCalone(noPI) 3. CellsstainedwithPIalone(noAnnexinV-FITC) PE 1. Unstainedcells 2. CellsstainedwithAnnexinV-PEalone(no7-AAD) 3. Cellsstainedwith7-AADalone(noAnnexinV-PE) Biotin 1. Unstainedcells 2. CellsstainedwithSAv-FITCalone(noAnnexinV-BiotinorPI) 3. CellsstainedwithAnnexinV-BiotinandSAv-FITC(noPI) 4. CellsstainedwithPIandSAv-FITC(noAnnexinV-Biotin) OtherStainingControls: Acelllinethatcanbeeasilyinducedtoundergoapoptosisshouldbeusedtoobtainpositivecontrolstaining.Itisimportanttonotethatthebasallevelofapoptosisandnecrosisvariesconsiderablywithinapopulation.Thus,evenintheabsenceofinducedapoptosis,mostcellpopulationswillcontainaminorpercentageofcellsthatarepositiveforapoptosis(AnnexinVpositive,vitaldyenegative).Theuntreatedpopulationisusedtodefinethebasallevelofapoptoticanddeadcells.Thepercentageofcellsthathavebeeninducedtoundergoapoptosisisthendeterminedbysubtractingthepercentageofapoptoticcellsintheuntreatedfromthetreatedpopulation. AnnexinVBlocking AnadditionalcontrolwhichmaybeperformedincludespreincubationofcellsampleswithrecombinantAnnexinV(unconjugated).ThisservestoblockAnnexinV-FITCbindingsitesandthusdemonstratesthespecificityofAnnexinV-FITCstaining.Theprocedurefollows. 1. WashcellstwicewithcoldPBSandthenresuspendcellsin1XBindingBufferataconcentrationof~1x106cells/ml. 2. Transfer100µlofthesolution(~1x105cells)toa5mlculturetube. 3. Add5-15µgofpurifiedrecombinantAnnexinV.TheamountofpurifiedrecombinantAnnexinVrequiredtosaturatebindingsitesmayvaryaccordingtocelltype,andstageofapoptosis.Insomecases,investigatorsmayalsoneedtoreducethenumberofcellsto0.5x105/100µlandstilladd5-15µgofrecombinantAnnexinVtoobtainoptimalresults. 4. Gentlymixthecellsandincubatefor15minatRT. 5. Add5µlofAnnexinV-FITC,AnnexinV-PEor*AnnexinV-Biotin.GentlymixandincubateatRTfor15mininthedark. *ForAnnexinV-Biotinsamplesonly:After15minincubation,washoncewith1mlof1XBindingBuffer.Dilute0.5µgStreptavidin-FluoresceinIsothiocynate(SAv-FITC,Cat.No.13024D)into100µlof1XBindingBufferandaddtothecellpellet.GentlymixthecellsandincubateatRTfor15mininthedark. 6. Add400µlof1XBindingBuffertoeachtube.Analyzebyflowcytometryassoonaspossible(within1hr).
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