BD流式细胞仪细胞周期分析艺术

1、Copyright2004NathanRegimbalCopyright2004NathanRegimbalCELLCYCLEANALYSISDYEFLUORESCENCEAnalysisofacellpopulationsstateofreplicationcanbeachievedbyfluorescentlylabelingthecellnucleithenanalyzingthefluorescencepropertiesofeachcellinthepopulationbyflowcytometry.QuiescentandG1cellswillhaveonecopyofDNAand

2、willthereforehave1Xfluorescenceintensity.CellsinG2/MphaseofthecellcyclewillhavetwocopiesofDNAandaccordinglywillhave2Xintensity.SincethecellsinSphasearesynthesizingDNAtheywillhavefluorescencevaluesbetweenthe1Xand2Xpopulations.DYEFLUORESCENCEAnalysisofacellpopulationsstateofreplicationcanbeachievedbyf

3、luorescentlylabelingthecellnucleithenanalyzingthefluorescencepropertiesofeachcellinthepopulationbyflowcytometry.QuiescentandG1cellswillhaveonecopyofDNAandwillthereforehave1Xfluorescenceintensity.CellsinG2/MphaseofthecellcyclewillhavetwocopiesofDNAandaccordinglywillhave2Xintensity.SincethecellsinSpha

4、searesynthesizingDNAtheywillhavefluorescencevaluesbetweenthe1Xand2Xpopulations.Copyright2004NathanRegimbalCopyright2004NathanRegimbalCopyright2004NathanRegimbalCopyright2004NathanRegimbalTheresultinghistrogramconsistsofthreepopulations:twoGaussiancurves(IXand2Xpeaks)andtheS-phasepopulation.Adjacentp

5、opulationsoverlapeachother.Becauseofthis,amodelingprogramisrequiredtode-convolutethepopulationsandassignpercentagevaluestoeachpopulation.Expertandsubjectivereviewofthemodelingsoftwarescellcyclephasepercentageassignmentisthefinalstageofcellcycleanalysispriortoreportingtheresults.G0G1:33.94%Mean:49.19

6、CV:2.97%G2M:15.28%Mean:96.41G2/G1:1.96S-Phase:N-FL2-WN-FL2-WFLOWCYTOMETRYANDCELLCYCLEDATA:DUEDILIGENCESpecialconsiderationsmustbetakenwhenoptimizingaflowcytometerforcellcycleanalysis.BecausetheDNAhistogramisthefinalproductoftheflowcytometerthatissubjectedtocurvede-convolutionanalysis,theintegrityoft

7、hedatamustbehighandthefidelityofindividualcellsfluorescencemustbemaintained.Thisismentionedbecauseofthesimplenatureofcellcycleanalysis:lookingateventswithdistinctfluorescencelevels.Asmentionedbefore,cellsinG0/1have1XfluorescenceandG2/Mcellshave2Xfluorescence.WhathappensiftwoG0/1cellspassthroughthebe

8、am(whetherstucktogetherornot)atthesametime?AGGREGATES:ASTICKYSITUATIONAsweallknow,cellscansticktoeachother.Also,despitethehydrodynamicfocusingthatoccursattheflowcytometerslaserintercept,multiplecellscanpassthroughthelaseratthesametimeeveniftheyrenotstucktogether.Whatresultsistherecordingofonetypeofe

9、ventasanothertypeentirely.Becauseyouareexaminingthenuclearfluorescenceofeachevent,twocellsinG0/1thatarestucktogetherwillhaveasmuchnuclearfluorescenceasoneG2/Mcell.ThiswillresultinanalyzedG2/Mpercentagesthatareerroneouslyhigh.OneG2CellTwoG1CellsOneG2CellTwoG1CellsCopyright2004NathanRegimbalCopyright2

10、004NathanRegimbalAggregatesandsimultaneouslyreadeventsareunavoidableduringtheacquisitionpartofcellcycleanalysis.Thewaytomaintainthefidelityofthedataistoexcludethesenon-singlecellsfromlateranalysis.Thisisaccomplishedbyusingthecytometerssignalpulseprocessingandtakingcaretooptimizetheinstrumentproperly

11、.PULSEPROCESSINGAscellstransitthroughthelaserbeam,theirfluorescencesignalsultimatelygeneratevoltagepulsesbythefluorescencedetector(photomultipliertube(PMT).Height=maximumfluorescenceintensity.Width=transittime.Area=totalfluorescenceofparticle.Ascellstransitthroughthelaserbeam,theirfluorescencesignal

12、sultimatelygeneratevoltagepulsesbythefluorescencedetector(photomultipliertube(PMT).Height=maximumfluorescenceintensity.Width=transittime.Area=totalfluorescenceofparticle.VIEWINGPULSEPROCESSINGPARAMETERSUsually,onlytheheight(maximumfluorescenceemission)signalisrecorded(soTHATSwhattheHinFL2-Hmeans一HEI

13、GHT!)Incellcycleanalyisis,however,allthreeparameters(H,WandA)mustbeaccountedfor.Pulsewidthisindicativeofparticletransittime.Justasatractortrailertakeslongertocrossanintersectionthandoesacompactcar,singlecellswillhavesmallerpulsewidthscomparedtocellsthathaveaggregated.BelowisahistogramoftheFL2peakemi

14、ssionvalues(FL2-H).Alone,itcanactasthesubjectofadeconvolutionprogram.However,sinceFL2-Halonegivesnoinformationabouttotalfluorescenceorsingularity,itcannotbeusedforaccuratecellcycleanalysis.091900SKWCCARR.O20HistogramsgeneratedwithCellQuestTMHistogramsgeneratedwithCellQuestTMProfromBectonDickinsonVi7

15、02004006008001000ksVERYsimilartotheFL2-Hlysis,itstilldoesnotallowforThisisahistogramofFL2-Area(totafcellfluorescence).Thisgraphlochistogram.AlthoughitisshowingusthecorrectparameterforDNAanaaggregatediscrimination.ksVERYsimilartotheFL2-Hlysis,itstilldoesnotallowfor091900SKWCCARR.020BETTER200400600FL2

16、-A8001000200400600FL2-A8001000THERIGHTWAYTOEXCLUDEAGGREGATES:FL2-Wvs.ATheleftplot(below)showspulsewidthversusarea,andthisistheplotusedtodistinguishbetweensinglecellsandaggregates.Singlecells(G0/1orG2/M)willhavesimilarpulsewidth(transittime)values.Aggregateswillhavelargerwidthvaluesandcanbeeasilyseen

17、ontheplottotherightofthesinglecellregion.Singlecellshavebeengated(leftplot)andanFL2-Areahistogramhasbeendrawnandformattedtoshowonlytheeventsinsideofthesinglecellregion.091900SKWCCARR.02002004006008001000FL2-A091900SKWCCARR.02002004006008001000FL2-ACopyright2004NathanRegimbalCopyright2004NathanRegimb

18、alCopyright2004NathanRegimbalCopyright2004NathanRegimbalThesearetheplotsyouwilluseintheanalysisprogram.Butbeforeyougettothatpoint,youmustfirstoptimizetheflowcytometertogeneratedatathatlookslikethis.Howisthisdone?AREVIEWOFCYTO-ANATOMY:PMTVOLTAGEANDAMPLIFIERWhenacellpassesthroughthelaser,apulseisgener

19、ated(seeabove).Assumingproperthresholdadjustment,thecharacteristicsofthisrawpulsewillbedeterminedbyonething:thecellandthePMTvoltage.LowVPMTpulsetHigherVLowVPMTpulsetHigherVPMTpulseByincreasingthePMTvoltage,youcanchangeapulsesheight,widthandareavalues.WHATANAMPGAINDOESSometimesyouneedtomovethingsarou

20、nddelicatelyonaparameter.YoudoitallthetimewithForwardScatter.EvernoticethattheFSCVoltageisadjustableonlyinlargeincrementsof10(E01,E00,E01,etc)?InordertocarefullymoveapopulationonForwardscatter,youneedtotakethatroughvoltagepulsefromtheFSCdetectorandchangeitslightly.ThisisdonewiththeFSCAmpgain.Notetha

21、ttheforwardscatterparameterislabeledFSC-H.TheHstandsforHeight.Theforwardscatterparameterisshowingonlytheheightofthevoltagepulse.Whattheampgaindoesisthis:ittakestherawvoltagepulsefromthedetectorandpre-amp(moremachineguts)anditcarefullyamplifiesthatsignalevenfurther.Inpracticality,theampgainonlyaffect

22、sapulsesHeightvalue.NoAmpGain:RawpulsefromPMTAmplified2xoriginal(ampreadout2.0)NoAmpGain:RawpulsefromPMTAmplified2xoriginal(ampreadout2.0)Copyright2004NathanRegimbalCopyright2004NathanRegimbalCopyright2004NathanRegimbalCopyright2004NathanRegimbal2HoutputSignalSIGNAL2HoutputSignalSIGNALIncreasetheamp

23、gainto2.0DoingsowilldoubletheheightoftherawvoltageHPulseWHITENOISETheampgainonaflowcytometerrangesfrom1.00(noalterationofrawvoltagepulse)to9.99(nearly10timestheamplification).OnFSC,ifyouneedtoamplifymorethan9.99,youadjusttheroughvoltagesettingtothenextstep(aten-foldincrease)andbringyourFSCAmpgainset

24、tingbackdownto1.00一andthenadjustyourampgainasnecessary.Anillustrationofamplifierfunctioninvolveselectronicmusicplayback.ACDplayerreadsthemediaandhasline-level(lowvoltage)outputsintheback.WhycantyousimplyconnectspeakersdirectlytoaCDplayer?Althoughthesignalispresentinhighfidelity,theamplitudeofthesign

25、alisverysmall.Sosmall,infact,thataspeakersmagnetwontbeenergizedwithenoughelectricitytovibrateandproducesound.WhatyouneedtodoisplugtheCDplayerintoanaudioamplifier.Theamplifiertakestheoriginalsignal(read,PMTvoltagepulse)andcarefullyincreasespulsesamplitudeateverypointofthesignalswaveform(read,ampgain)

26、.Theresultingsignal,havingmuchhigheramplitude,willnowhaveenough“oomph”topoweraloudspeaker.Inflowcytometry,wedontneedtoaddsomuchampgaintoourPMTvoltagepulsethatwecanpoweraloudspeaker,butwedoaddenoughampgaintoinchituponaparametertomakeitlookgood.SOWHATDOIADJUSTTOOPTIMIZEFORCELLCYCLEANALYSIS?Stepbystepo

27、ptimizationusingCellQuest-besuretoaskforexpertassistance:Drawthefollowingplots:Twoparameter:FSC/SSC,FLW/FL2A;Histograms:FL2-H,FL2-ASetyourthresholdonFL2at20.Intheartofflowcytometriccellcycleanalysis,itisstandardtosetthethresholdontheDNAfluorescenceparameteratavalueof10%ofthelocationoftheG0/1peak.The

28、G0/1peakwillthereforebeplacedatavalueof200ontheFL2-Hparameter(seebelow).SelectFL2astheDDMparameter(lowerrightportionoftheDetectors/AmpGainwindowinCQ).PutFL2inLINamplification.Linearamplificationisnecessarytoresolvethe1xand2xfluorescencepeaksoftheG0/1andG2/Mcells(respectively)ontheFL2parameter.Ifthea

29、mplificationwerelogarithmic,thepeakswouldbesmashedtogether.PlacetheAmpgainsforFL2,FL2-AandFL2-Wat1.00.PlaceasampleonthemachineandruninLOWspeed(lowspeedensureslowestpossibleCVsandminimizescoincidenceevents)BecausethethresholdissetonFL2,noeventswillbeseenunlesstheFL2PMThasenoughvoltagetogeneratepulses

30、abovethreshold.IncreasetheFL2voltageifno/feweventsareregisteringinthemachine.Oncetheeventsareabovethreshold,adjustFSCandSSCasusual.TheG0/1peakwillbediscernablefromtheG2/Mpeak.Withadiploidcontrol,youwillseetwodistinctpeaksontheFL2-Hhistogram.AdjusttheFL2voltagesothattheG0/1peakhasavalueofabout100onFL

31、2-H.ThereshouldbearoughlysimilarprofileatthispointontheFL2-Ahistogram.IncreasetheFL2ampgain(notvoltage)tomovethepeaksupontheFL2-Hhistogram.Sincetheampgainonlyaddsheighttoarawvoltagepulse,thepopulationwillonlymoveonFL2-H.TheotherFL2derivedparameters(WandA)willbeunaffected.IncreasetheFL2-Aampgainuntil

32、theG0/1peakhasavalueof200onFL2-AIncreasetheFL2-Wampgainuntilthesinglecellpopulationhasavaluebetween200and600onFL2-W.AdjusttheFL2voltage,ampgainandtheFL2-WandAampgainsuntilthedataappearssimilartothedatabelow:Copyright2004NathanRegimbalCopyright2004NathanRegimbalWHATGOODDATALOOKSLIKEALALCopyright2004N

33、athanRegimbalCopyright2004NathanRegimbalCopyright2004NathanRegimbalCopyright2004NathanRegimbalGeneratingdatalikethisinvolvescarefuladjustmentsandreadjustmentsofPMTvoltageandAmpgainsettings.ACQUISITIONDETAILSAccuratede-convolutionoftheDNAhistogramrequiresthatatleast10,000eventsareavailableforanalysis

34、.Becausethesinglecellsarethesubjectofanalysis,itsimportantthat,duringacquisitionsetup,acollectiongateisusedtoensurethatenoughsinglecellswillbestored.Thebestplotforaggregate/debrisdiscriminationistheFL2-Wvs.FL2-Aplot.Usethisplottodrawagatearoundyoursinglecells.Thefollowingplotshowsthesinglecellgateaswellastheotherpopulationsontheplot.、._jLi-13DEBRIS/DIPLOIDCONTINUUMAGGREGATESDEBRISSINGLECELLS091900SKWCCARR.02002004006008001000FL2-WSetAcquisitionandStoragesothat10,000eventsarecountedin