Materials

• Thefluorochrome-conjugatedantibodyorantibodiesofinterest.
• AnappropriateDNAbindingdye.Forsinglephenotypeanalysis,propidiumiodidecanbeusedsimultaneouslywithFITC-conjugatedantibodiesforanalysisonasinglelaserflowcytometer.AnalysisoftwophenotypesonasinglelaserinstrumentcanbeaccomplishedusingFITC-andPE-conjugatedantibodiesandthefarredDNAbindingdyes7-aminoactinomycinDorLDS-751.Hoechst33342and33258orDAPIcanbeusedonduallaser(488nm/UV)instrumentsforsimultaneousanalysiswithtwoorthreefluorochrome-conjugatedantibodies.RNaseshouldbeaddedforDNAdyesthatcanbinddsRNA(suchaspropidiumiodide).
• 70%EtOHinddH₂0
• 50%fetalbovineserum(FBS)inPBSwith0.1%sodiumazide
• 70%EtOHinglycerol
• stainingbuffer(2%FBSinPBSwith0.1%sodiumazide)

Procedure

• PreparethecelltypeofinterestasasinglecellsuspensionandwashoncewithcoldPBSwith0.1%sodiumazide.Labelwiththefluorochrome-conjugatedantibodyofinterestincoldstainingbuffer.
• WashcellsoncewithstainingbufferandoncewithPBS/azidealone.Decantthesupernatant,shaketubegentlytoresuspendpelletandadd1part(i.e.0.3mls)50%FBSinPBS.Whilegentlymixing,add3parts(i.e.0.9mls)cold70%EtOHinddH₂0dropwise.SomeprecipitationoftheFBSproteinswilloccurduringadditionofethanol;thiscanbedisregarded,astheseprecipitateswillberemovedduringsubsequentwashes.Incubateforatleasttwohoursorovernightat4^oC.
• WashthecellstwicewithcoldPBS/azidetoremoveEtOHandprecipitatedproteinandaddpropidiumiodideat50mg/mlinPBSwith100U/mlDNase-freeRNase.OtherDNAcontentdyescanbesubstitutedforpropidiumiodideatthispoint.7-aminoactinomycinDcanbeusedatconcentrationsbetween10and25mg/ml,andHoechst33342and3258andDAPIat1to2mg/ml,forexample.Analyzeusingtheappropriateinstrument.
• Preliminaryevidencesuggeststhat70%EtOHinglycerolcanbesubstitutedfor50%FBSfollowedby70%EtOHadditionwithsimilarpreservationofsurfacemarkers.
References

Fraker,P.J.,King,L.E.,Lill-Elghanian,D.andTelford,W.G.(1994)Quantitationofapoptoticeventsinpureandheterogeneouspopulationsofcellsusingflowcytometry.InMethodsinCellBIOLOGyVolume46:CellDeath,B.OsborneandL.Schwartz,eds.,AcademicPress,NewYork,NY,pp.57-76.

Telford,W.G.,King,L.E.andFraker,P.J.(1994)Rapidquantitationofapoptosisinpureandheterogeneouscellpopulationsusingflowcytometry(reviewarticle).JournalofImmunologicalMethods172,1-16.

Telford,W.G.andFraker,P.J.(1995)PreferentialinductionofapoptosisinmouseCD4+CD8+TCRloCD3lothymocytesbyzinc.JournalofCellularPhysiology164,259-270.

Garvy,B.A.,Telford,W.G.,King,L.E.andFraker,P.J.(1993)GlucocorticoidsandirradiationinducedapoptosisinnormalmurinebonemarrowB-lineagelymphocytesasdeterminedbyflowcytometry.Immunology79,270-277.

Telford,W.G.,King,L.E.andFraker,P.J.(1992)ComparativeevaluationofseveralDNAbindingdyesinthedetectionofapoptosis-associatedchromatindegradationbyflowcytometry.Cytometry13,137-143.

Telford,W.G.,King,L.E.andFraker,P.J.(1991)Evaluationofglucocorticoid-inducedDNAfragmentationinmousethymocytesbyflowcytometry.CellProliferation24,447-459.