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CELL CYCLE ANALYSIS

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PROPIDIUMIODIDE:ThemostcommonlyuseddyeforDNAcontent/cellcycleanalysisisPROPIDIUMIODIDE(PI).Itcanbeusedtostainwholecellsorisolatednuclei.ThePIintercalatesintothemajorgrooveofdouble-strandedDNAandproducesahighlyfluorescentadductthatcanbeexcitedat488nmwithabroademissioncentredaround600nm.SincePIcanalsobindtodouble-strandedRNA,itisnecessarytotreatthecellswithRNaseforoptimalDNAresolution.TheexcitationofPIat488nmfacilitatesitsuseonthebenchtopcytomters.However,PIcanalsobeexcitedintheU.V.(351-364nmlinefromtheargonlaser)whichshouldbeconsideredwhenperformingmulticolouranalysisonthemultibeamcellsorters.

ProtocolforstainingwholecellswithPI:

1.HarvestcellsandpreparesinglecellsUSPensioninbuffer(e.g.PBS+2%FBS;PBS+0.1%BSA)2.WashcellsX2andresuspendat1-2x106cells/ml.3.Aliquot1mlcellsina15mlpolypropylene,V-bottomedtubeandadd3mlcoldabsoluteethanol.(TheethanolcanbeaddedforcIBLybyexpellingfromaPipetteordropwisewhilevortexing…determinethebestmethodforeachcelltypetopreventclumpingandcellloss.)4.Fixcellsforatleast1hourat4oC.(Cellsmaybestoredin70%ethanolat-20oCforseveralweekspriortoPIstainingandflowcytometricanalysis).5.WashcellsX2inPBS.(Itmaybenecessarytocentrifugecellsataslightlyhigher"g"topelletafterethanolfixationasthecellsbecomefloculent.)6.Add1mlofpropidiumiodidestainingsolutiontocellpelletandmixwell.Add50ulofRNaseAstocksolutionandincubate3hrat4oC.7.Storesamplesat4oCuntilanalysedbyflowcytometry.

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REAGENTS:PropidiumIodideStainingSolution:3.8mMsodiumcitrate,50ug/mlPI[Sigma,P4170]inPBS.RNaseAstocksolution:10ug/mlRNaseA[WorthingtonBiochemicals,RASELS005649,LS005650](boiledfor5min,aliquotedandstoredfrozenat-20oC).

References:

CrissmanHA,SteinkampJA.RapidsimultaneousmeasurementofDNA,proteinandcellvolumeinsinglecellsfromlargemammaliancellpopulations.J.CellBiol.,59:766,1973.

KrishanA.Rapidflowcytofluorometricanalysisofcellcyclebypropidiumiodidestaining.J.CellBiol.,66:188,1975.

ACRIDINEORANGE:DifferentialStainingofDNA/RNAwithACRIDINEORANGE(AO)canbeperformedforsimultaneousassessmentofDNAandRNAcontentofcells.TheAOexhibitsdifferentspectralcharacteristicswhenboundtoDNAorRNAthatcanbeexcitedat488nmwitheitheraGREENorREDfluorescencerespectively.

ProtocolforstainingwholecellswithAO:

1.WashcellsX2inbuffer(e.g.PBS+2%FBS;PBS+0.1%BSA).2.Resuspendcellsto1x106cells/mlinbuffer.3.Add200mltoacleantesttubeandadd400mlofSolutionA.Mixgentlybyswirlingtube.(DONOTVORTEX).4.Standfor15secondsandthenadd1.2mlofice-coldSolutionB.5.Standonicefor5to15mintoallowsampletoequilibrateandanalyzeforfluorescenceimmediately.

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REAGENTS:SolutionA:0.1%TritonX-100,0.08NHCl,0.15MNaClSolutionB:6ug/mlAcridineOrange[Sigma,A6014],1mMEDTA-Na,0.15MNaCl,0.2MNaPO4,0.1MCitricacidbuffer,pH6.0

References:TraganosF,DarzynkiewiczZ,SharplessTetal.Simultaneousstainingofribonucleicanddeoxyribonucleicacidsinunfixedcellsusingacridineorangeinaflowcytofluorometricsystem.J.Histochem.Cytochem.,25:46,1977.

DarzynkiewiczZ,TraganosF,MelamedMR.Newcellcyclecompartmentsidentifiedbymultiparameterflowcytometry.Cytometry,1:98,1980.

HOECHST:TheHoechstdyes33342and33258arebis-benzimidederivatives.Theycanbeusedforcellcycleanalysisofviablecellsandcanbeusedatlowconcentrationslimitingtoxicityproblems.TheHoechstdyesbindtoAT-richregionsoftheDNAandwhenexcitedwithaU.V.source(e.g..(351-364nmlinefromtheargonlaser)producebrightfluorescenceat465nm.DAPI(diamidino-2-phenylindole2HCl)isalsoanATbinderwithsimilarspectralpropertiestotheHoechstdyes.

ProtocolforstainingwholecellswithHOECHST:

1.WashcellsX2inbuffer(e.g.PBS+2%FBS;PBS+0.1%BSA).2.Resuspendcellsto1x106cells/mlinbuffer.3.AddanequalvolumeofHoechstdye(workingsolution5mM).4.Incubatefor15minat37oC.5.Analyseforfluorescenceorcellscanbewashedandfixedinparaformaldehyde(1%inPBS)forfutureanalysis.

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REAGENTS:StockSolution:Dissolve2mgofHoechst33342[vendor,catno.]in1mlofDMSO.Storeat4oC.WorkingSolution:Add1.4mlofstocksolutionto1mlPBS.

References:LoeweH,UrbanietzJ.Arnzeimforsch,24:1927,1974.

LattSA.Microfluorometricdetectionofdeoxyribonucleicacidreplicationinhumanmetaphasechromosomes.Proc.Natl.Acad.Sci.USA.70:3395,1973.

LattSA,StettenG.Spectralstudieson33258Hoechstandrelatedbisbenzimidazoledyesusefulforfluorescentdetectionofdeoxyribonucleicacidsynthesis.J.Histochem.Cytochem.,24:24,1976.

BiglerRD.Acomparisonoflow-powerhelium-cadmiumandargonultravioletlasersincommercialflowcytometers.Cytometry,8:441,1987.

ShapiroHM.FlowcytometricestimationofDNAandRNAcontentinintactcellsstainedwithHoechst33342andpyroninY.Cytometry,2:143,1981.

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CELL&nbsp;CYCLE&nbsp;ANALYSIS

CELL CYCLE ANALYSIS