Analysis of Cell Cycle Position in Mammalian Cells.

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by  Cecchini, Matthew J.

JournalofVisualizedExperiments www.jove.com
VideoArticle
Analysis of Cell Cycle Position in Mammalian Cells
MatthewJ. Cecchini1,Mehdi Amiri1,FrederickA. Dick2
1DepartmentofBiochemistry,SchulichSchoolofMedicineandDentistry,UniversityofWesternOntario
2LondonRegionalCancerProgram,Children'sHealthResearchInstitute,andDepartmentofBiochemistry,SchulichSchoolofMedicineandDentistry,Universityof
WesternOntario
URL:http://www.jove.com/video/3491/
DOI:10.3791/3491
Keywords:MolecularBiology,Issue59,cellcycle,proliferation,flowcytometry,DNAsynthesis,fluorescence,
DatePublished:1/21/2012
Citation:Cecchini, M.J.,Amiri, M.,Dick, F.A. AnalysisofCellCyclePositioninMammalianCells.J.Vis.Exp.(59),e3491,DOI:10.3791/3491
(2012).
Abstract
Theregulationofcellproliferationiscentraltotissuemorphogenesisduringthedevelopmentofmulticellularorganisms.Furthermore,lossof
controlofcellproliferationunderliesthepathologyofdiseaseslikecancer.Assuchthereisgreatneedtobeabletoinvestigatecellproliferation
andquantitatetheproportionofcellsineachphaseofthecellcycle.Itisalsoofvitalimportancetoindistinguishablyidentifycellsthatare
replicatingtheirDNAwithinalargerpopulation.Sinceacell′sdecisiontoproliferateismadeintheG1phaseimmediatelybeforeinitiatingDNA
synthesisandprogressingthroughtherestofthecellcycle,detectionofDNAsynthesisatthisstageallowsforanunambiguousdeterminationof
thestatusofgrowthregulationincellcultureexperiments.
DNAcontentincellscanbereadilyquantitatedbyflowcytometryofcellsstainedwithpropidiumiodide,afluorescentDNAintercalatingdye.
Similarly,activeDNAsynthesiscanbequantitatedbyculturingcellsinthepresenceofradioactivethymidine,harvestingthecells,andmeasuring
theincorporationofradioactivityintoanacidinsolublefraction.Wehaveconsiderableexpertisewithcellcycleanalysisandrecommenda
differentapproach.WeInvestigatecellproliferationusingbromodeoxyuridine/fluorodeoxyuridine(abbreviatedsimplyasBrdU)stainingthat
detectstheincorporationofthesethymineanalogsintorecentlysynthesizedDNA.LabelingandstainingcellswithBrdU,combinedwithtotalDNA
stainingbypropidiumiodideandanalysisbyflowcytometry1offersthemostaccuratemeasureofcellsinthevariousstagesofthecellcycle.Itis
ourpreferredmethodbecauseitcombinesthedetectionofactiveDNAsynthesis,throughantibodybasedstainingofBrdU,withtotalDNA
contentfrompropidiumiodide.ThisallowsfortheclearseparationofcellsinG1fromearlySphase,orlateSphasefromG2/M.Furthermore,this
approachcanbeutilizedtoinvestigatetheeffectsofmanydifferentcellstimuliandpharmacologicagentsontheregulationofprogression
throughthesedifferentcellcyclephases.
Inthisreportwedescribemethodsforlabelingandstainingculturedcells,aswellastheiranalysisbyflowcytometry.Wealsoinclude
experimentalexamplesofhowthismethodcanbeusedtomeasuretheeffectsofgrowthinhibitingsignalsfromcytokinessuchasTGF-β1,and
proliferativeinhibitorssuchasthecyclindependentkinaseinhibitor,p27KIP1.Wealsoincludeanalternateprotocolthatallowsfortheanalysisof
cellcyclepositioninasub-populationofcellswithinalargerculture5.Inthiscase,wedemonstratehowtodetectacellcyclearrestincells
transfectedwiththeretinoblastomageneevenwhengreatlyoutnumberedbyuntransfectedcellsinthesameculture.Theseexamplesillustrate
themanywaysthatDNAstainingandflowcytometrycanbeutilizedandadaptedtoinvestigatefundamentalquestionsofmammaliancellcycle
control.
VideoLink
Thevideocomponentofthisarticlecanbefoundathttp://www.jove.com/video/3491/
Protocol
1. Labeling and fixing of cells
1. Add1μLofCellProliferationLabelingReagent(BrdU)permLofcellculturemedium(1to1000dilution)1hourbeforeharvesting.The
labelingperiodmayneedtobelengthenedforslowergrowingcells.
2. Toharvestcells,aspirateculturemediumandwashthoroughlywithphosphatebufferedsaline(PBS).Repeattothoroughlyremovetracesof
medium.
3. WashculturesquicklyathirdtimewithPBScontaining3mMEDTAandaspiratethoroughly.
4. AddasmallvolumeofPBScontaining3mMEDTAtoeachdish,0.5mLfora6cmdishisideal.Incubateat22°Cforapproximately5minutes
todetachcells.Transfertoa15mLconicaltube.
5. Centrifugecellsat500xgfor5minutestopellet,removesupernatant,andresuspendthoroughlyin100μLofPBS.
6. Fixcellsbyadding5mLof95%EtOH,dropwise,whilevortexing.Atthisstep,cellscanbestoredat4°Cforatleastamonth.
2. Denaturing and staining of BrdU and DNA
1. Centrifugecellsat500xgfor5minutestopelletcellsandremove95%EtOH.Resuspendin1mLof2NHCland0.5%Tx-100byaddingina
dropwisefashionwhilevortexing.Incubateatroomtemperaturefor30minutes.
2. Centrifugeasbeforeinsection2.1andcarefullyaspiratesupernatantsincethecellsformaveryloosepelletatthisstep.Gentlyresuspendin
1mLof0.1MNaB4O7(pH8.5)andincubatedforatleast30minutesatroomtemperature.
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3. Pelletcellsasaboveinsection2.1andresuspendin0.5mLofantibodysolution(PBScontaining1%BSAand0.2%Tween-20)withmouse
anti-BrdUantibodiesdiluted1to50.Incubateatroomtemperaturefor30minutes.
4. Pelletcellsagainandresuspendin50mLofantibodysolutioncontainingrabbitanti-mousesecondaryantibodiesconjugatedtoFITCdiluted
1to25.Incubatefor30minutesonbenchtopandprotectfromlight.
5. Centrifugecellsafinaltimeandresuspendin0.5mLofpropidiumiodideandRNasesolution(PI-RNAsesolution,PBSwith1%BSA,10
mg/mLpropidiumiodide,0.25mg/mLRNaseA)andincubateinthedarkat37°Cfor30minutes.AlternativelyRNAcanbedigestedovernight
at4°Cinthedark.
6. Passsolutionthroughacellstrainertoremoveaggregatesandcollectsinglecellsinanappropriatetubeforsampleuptakeonyourflow
cytometer.Again,samplesshouldbeprotectedfromdirectexposuretolight.
3.Analysis by flow cytometry
1. Cellsshouldbeanalyzedbyastandardflowcytometerthatiscapableofdiscriminatingagainstdoublets(twocellsthatpassthroughtheflow
cellasone)withappropriatedetectioncapabilityforpropidiumiodideandFITC.Singlecellscanbedetectedinthisflowcytometryapplication
bygatingforeventsbasedonforwardscatterandsidescatterpopulations,andbyusingthepropertiesofDNAstainingbypropidiumiodide.
Theappearanceofadoubletdiscriminatingdotplotvariesbetweenflowcytometersandreliesonpropidiumiodidestainingproperties,see
yourlocalflowcytometryfacilityforadviceonsettingupadoubletdiscriminatorinyouranalysis.Ingeneral,inanasynchronouslyproliferating
populationofcells,singlecellsareusuallythetwomostabundantpeaks(2Nand4NDNA)inthedoubletdiscriminatingplotandagate
shouldbedrawnaroundthem.Thiswillselectsinglecelleventsthatareusedforallsubsequentanalysisbelow.
2. Thefirstsampletobeanalyzedshouldbeanasynchronouslyproliferatingculturethatservesasapositivecontrolforstainingandflow
cytometerset-up.Adjustthesensitivityofphotomultipliertubesforpropidiumiodidestainingsuchthatthe2Nand4Npeaksfromsingletcells
arecenteredat200and400(arbitraryunits)ontheX-axis.Weoftenstainanabundantsupplyofthesecellstoensurethatallparametersof
theflowcytometerhavebeenadjustedappropriatelywithoutusingupthissample.Thispositivecontrolisalsohelpfulfornewusersto
becomemorefamiliarwiththeoperationoftheflowcytometer.
3. AdjustthesensitivityofthephotomultipliertubeforFITCdetectionsuchthatG1andG2/Mpopulationsareabovebackground.BrdUpositive
cellsshouldbeapproximately10timesbrighterandtheY-axisshouldbedisplayedasalogarithmicscale.Itissometimeshelpfultoincludea
negativecontrolforBrdUstaining(byreplacingtheanti-BrdUantibodieswithnon-specificIgGinstep2.3above)toclearlydistinguish
positivelystainedcells.
4. AdjustcompensationbetweenpropidiumiodideandFITCsuchthatsingleeventscapturedbytheflowcytometerformahorseshoeshaped
arcfromG1inthelowerleftuptoS-phaseanddownintothelowerrightforG2/M.Pleaseseethefollowingreferenceforamoreindepth
discussionoftheprinciplesanduseofflowcytometryandreferencesthereinforspecificapplications2.
4. Data analysis
1. 5000to10000singlecelleventswiththedesiredrangeofDNAcontentshouldbecollectedforeachsampleinordertohaveconfidencethat
thepopulationofcellsintheculturehavebeenthoroughlysampled.
2. OncecellshavebeenmeasuredforpropidiumiodideandBrdUcontenttheyneedtobeassignedtotheG1,S,orG2/Mphases.Dothisby
drawinggatesaroundthetwoBrdUnegativepopulationscenteredat200and400(G1andG2/Mrespectively).Everythingabovethese
boxesshouldbeincludedinasinglegatethatmeasuresS-phase(Fig.1A).Thepercentageofcellsineachgaterepresentstherelative
numberofcellsinG1,S,andG2/M(Fig.1B).
5.Alternate protocol for analysis of cell cycle in a mixed population of cells
1. Thisalternateprotocolismosthelpfulwhentransfectionofexpressionvectorsroutinelyresultsinalowpercentageofcellsexpressingthe
geneproductofinterest,orwhendrugtreatmenttoselectapurepopulationofcellsisimpractical.Thisresultsinarelativelysmallproportion
ofcellsofinterestbeingcontaminatedwithalargepopulationofuntransducedcells.Inthiscase,co-transfectplasmidsexpressingyourgene
orshRNAofinterestalongwithaplasmidthatexpressesamarkerfordetectingtransfectedcellsbyflowcytometry,suchasamembrane
anchoredGFP3,oraforeigncellsurfacemarkersuchasCD194orCD205.
2. ForthepurposesofthisprotocolwewilluseCD20stainingasanexample,however,stainingforCD19isessentiallyidentical.Inthecaseof
transfectionwithCD20,thereisnoneedtoBrdUlabel,simplyharvestcellsasdescribedinsteps1.2to1.5aboveandstainbyadding20μL
FITCconjugatedanti-CD20antibodiestothecellsonicefor20minutesinthedark.Fixcellsasdescribedinstep1.6.Cellscanagainbe
storedforatleastamonthat4°Cinthedark.FordetectionofGFP,omittheantibodystainingfromthisstepandfixandstorecellsas
described.
3. Re-hydratethecellsbypelletinginthecentrifugeat500xgfor5minutesandresuspendingin5mLofPBScontaining1%BSA.
4. Re-pelletthecellsat500xgfor5minutesandresuspendcellsin0.5mLofpropidiumiodideandRNasesolutionasdescribedabovein
section2.5.
5. Continuetofollowcellpreparationinstructionsin2.6andanalysisinstructionsin3.1and3.2.
6. AdjustthesensitivityofthephotomultipliertubeforFITCdetectionsuchthatunstainedcellsareabovebackground.Ideally,CD20-FITC
positivecellswillbe10Xto100XmoreintenselystainedthanbackgroundandtheY-axisofthisplotshouldbedisplayedasalogarithmic
scale(Fig.2A).CD20positivecellsthatare10Xbrighterthanbackground(andwithpropidiumiodidestainingbetween200and400)should
beselectedfordisplayinapropidiumiodideversuscellcountshistogramasshowninFig.2C.Forcelllinesthatexpressmarkersthatare
readilystainedbrightly(ie.10Xto100Xabovebackground)theCD20positivecut-offshouldbesetat10Xbackground.Inclusionofweaker
stainingcellsincreasesvariabilityintheseexperiments,presumablybecausethegeneofinterestisalsoweaklyexpressedinthesecells.For
celllinesthatonlyyieldweaklystainedCD20positives(ie.lessthan10Xbackground),determinationofacutoffshouldbemadeusinga
negativecontrolconsistingofCD20stained,untransfectedcells.
7. Atleast1000CD20positiveeventsshouldbecollectedforeachsample.Experimentswithfewerthat1000eventswillproducehigh
variability.SoftwarepackagessuchasModFitLT(VeritySoftware),MultiCycleAV(PhoenixFlowSystems),andFlowJo(TreeStar)use
mathematicalestimatesoftheG1,S,andG2/Mpopulationsthatcontributetotheshapeofthecurveinhistogramssuchasthoseshownin
Fig.2BandC.
6. Representative Results:
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Weprovidethreeexamplesofexperimentalcellcycleanalysesusingourapproaches.Thefirstusesretroviralexpressionofthecyclindependent
kinaseinhibitorp27KIP1inmouseembryonicfibroblasts.Twentyfourhoursafterdrugselectionforviralinfectionwascomplete,cellswerepulse
labeledwithBrdUforonehour.Inthisexperimentectopicexpressionoftheinhibitorisusedtoarrestproliferationofthecells(Fig.3AandB).As
showninFig.3BlittleBrdUpositiveeventsareevidentintheS-phasegateinresponsetop27.Likewise,thepercentageofcellsinS-phasefor
p27expressingcellsisquitelowasdiagramedinFig.3C.Thistypeofanalysishasbeenveryeffectiveincharacterizingthecellcyclecontrol
defectsincellsderivedfromvariousstrainsofgene-targetedmice6,7,8.
Inthesecondexperiment,untransformedmammaryepithelialcells(MCF10A)weretreatedwiththegrowthinhibitorycytokine,transforming
growthfactorbetaone(TGF-β1)for24hours.CellswerelabeledwithBrdUforfourhoursimmediatelypriortoharvesting.AsshowninFig.4
BrdUlabelinginS-phasecellsisgreatlydiminishedbyTGF-β1signaling,thespecificityofourstainingisalsovalidatedwithanIgGnegative
control(Fig.4C).QuantificationofthedifferentphasesofthecellcycleconfirmsthatTGF-β1primarilyinhibitsproliferationintheG1phaseofthe
cellcycle,leadingtoanaccumulationinthisphase.
Inourlastexample,pRBdeficientSaOS-2cellsaretransfectedwithaCMV-CD20expressionvectorandeitherCMV-RBorCMV-β-Galasa
control.Threedaysfollowingtransfectioncellswereharvested,stained,andfixed.FlowcytometryanalysisofthesecellsisshowninFig.5.This
demonstratesthecellcycledistributionofnegativecontroltransfectedcells(Fig.5A)incomparisonwiththedistributionfollowing72hoursofpRB
expression(Fig.5B).FollowingcurvefittingbyMultiCyclesoftware,adirectcomparisonoftheproportionofcellcyclephasesisshowninFig.
5C.ThisrevealstheaccumulationofcellsinG1followingpRBexpressionandtherelativedepletionofcellsfromtheSandG2/Mphases.We
haveusedvariationsonthisassaytoprobeG1arrestmechanismsextensively9,10,11,12,13.
Theseexamplesdemonstratehowcellcyclecontrolcanbemeasuredinresponsetoavarietyofstimuliorcellmanipulations.Thisapproachcan
thereforebeadaptedtomanyapplicationsthatrequirethemeasurementofcellcyclepositioninculturetypeexperiments.

Figure1.QuantitationofcellcyclephasesbycombinedpropidiumiodideandBrdUstaining(PI-BrdU).(A)Thispaneldisplaysthreedimensional
flowcytometryofpropidiumiodideandBrdUstainedcells.Notethatcellswith2Nand4NDNAcontentarecenteredoverthe200and400marks
ontheX-axisscaleforpropidiumiodidestainingintensity.BrdUstainingintensityismeasuredonalogarithmicscaleontheY-axis.Notethe
positionofgatesusedtoquantitatecellsintheG1,S,andG2/Mphasesofthecellcycle.(B)TherelativeproportionofcellsineachoftheG1,S,
andG2/MgatesfromAareshownonthisgraph.

Figure2.QuantitationofcellcyclephasesinselectedcellsusingpropidiumiodideandthecellsurfacemarkerCD20.(A)Thispanelshows
propidiumiodideandCD20stainingforamixtureofcells,someofwhichectopicallyexpressCD20andpRB.Notethatthecellswith2Nand4N
DNA(themostabundantpopulations)arecenteredoverthe200and400marksontheX-axis.ThepositionoftheCD20+gateselectscellsthat
arestainedatleast10Xmorebrightlythanbackground.(B)AgraphofcellcountsversuspropidiumiodidestainingisshownforCD20negative
cellsinpanelAthatareasynchronouslyproliferating.(C)AsimilargraphisshownforCD20positivecellsfrompanelAthathavebeeninducedto
arrestwithpRBexpressionandcontaincellswithprimarily2NDNAcontent.Thisdemonstratesthatanarrestedsub-populationcanbe
distinguishedfromothercellsinthiscultureusingthisstainingtechnique.
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Figure3.Inhibitionofcellproliferationbyp27KIP1.(A)PI-BrdUanalysisisusedtomeasurethecellcyclephasesinanasynchronously
proliferatingpopulationofcellsthatweretransducedwithanemptypBABEretroviralvector.(B)Asimilaranalysisofcellstransducedwith
pBABE-p27.NotetheabsenceofcellsintheS-phaseandgreaterintensityofeventsinG1andG2/Mgates.(C)Quantitationofcellsinthe
respectivephasesofthecellcycleinasynchronouslyproliferatingcontrolandp27expressingcells.
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Figure4.InhibitionofcellproliferationbyTGF-β1(A)PI-BrdUanalysisofasynchronouslyproliferatingMCF10Acells.(B)Analysisofcellstreated
with100pMofTGF-β1for24hours.NotetheaccumulationofcellsprimarilyintheG1phaseofthecellcycle.(C)ValidationofBrdUstainingby
replacingtheanti-BrdUprimaryantibodywithanon-specificIgGcontrolinasynchronouslyproliferatingcells.(D)Graphicalquantitationofthe
respectivecellcyclephasesfromAandB.
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Figure5.InhibitionofcellproliferationbypRB.(A)CellcountsversuspropidiumiodidestainingofCD20andß-galtransfectedcellsisshown.
Thisisanimportantcontrolastransfectioncanpartiallysynchronizecells,renderingtheuntransfectedpopulation(CD20-cells)asan
inappropriatecontrolforthetransfectedsub-population.Transfectedcellsneedtobecomparedwithotheranalogouslytransfectedcells.(B)Cell
countsversuspropidiumiodidegraphofCD20andpRBtransfectedcells.Notethealmostexclusivepresenceofa2Npeak.(C)Graphical
representationofcellcyclephaseproportionsdeterminedfromAandBusingcurvefittingmethodsinMultiCyclesoftware.
Discussion
Inourexperience,successwiththesetechniquesisdependentonafewkeycontrolsandexperimentalconditions.Oneisestablishingan
asynchronouslyproliferatingcontrolforuseintheseexperiments.Thiscontrolservesthreeimportantpurposes.First,itensuresthatculture
conditionsusedforallexperimentalsamplesareadequatetosupportcontinualproliferationintheabsenceoftreatment.Thissamplealsoserves
thepurposeofapositivecontrolforthestainingmethodologytoensurethatBrdUorCD20positivecellscanbedetectedwhentheyarepresent.
Lastly,thissampleisusedtocalibratetheflowcytometer.Thiscontrolsamplecanbeusedtoadjustpropidiumiodidestainingintensitytodetect
2Nand4Ncellsat200and400respectively.Furthermore,detectionsensitivityofBrdUorCD20canbeadjustedsothatnegativeandpositive
signalsarecenteredasshowninFigures1Aand2A.WhenallsampleshaveasimilarconcentrationofcellsinPI-RNasesolution,thenfew
adjustmentstothecytometerareneededassubsequentsamplesarerun.ThisisimportantforreasonsshowninFig.4Band5BwherestrongG1
accumulationcreatesessentiallyasingleG1peakthatcouldbemisinterpretedasG2/M.
Therepresentativeexperimentsalsomakeotherimportantpoints.Firstofall,theydemonstratethatBrdUuptakeandlabelingcanvarybetween
celltypes.Forthisreasonitisimportanttoempiricallydeterminethelengthofpulseandstainingconditionsneededtoadequatelydetectcellsin
S-phase.Ingeneral,celltypesthatdoubleinculturein24hoursorlesscanbelabeledwithBrdUinanhour.Slowergrowingcelltypesmay
requirelongerpulsesandalterationstoantibodystainingconditionsandduration.MCF10Acellsareanexcellentexampleinthisregardasthey
werelabeledwithBrdUforfourhoursandstainedwithtwicethestandardconcentrationofantibodiesforfourtimesaslong.Investigatorsshould
becarefulnottoexceed6hoursofBrdUlabelingevenwithveryslowlygrowingcellsasthiscanerroneouslyleadtoinclusionofG2/Mcellsinthe
S-phasepopulation.Secondly,incomparingtheeffectsofp27KIP1expressionwiththoseofTGF-β1,itisclearthatp27caninducean
accumulationineitherG1orG2/MphaseswhileTGF-β1signalinginducesaG1arrest.Traditionalmeansofdetectingproliferationsuchas3
H-Thymidineincorporationandscintillationcountingareunabletodistinguishthesepossibilities.
Inouralternateprotocolwedemonstratethedetectionofasub-populationofcellsinalargeruntransfectedpopulation.Itisimportantto
empiricallydeterminethemostappropriatereporterfordetectingtransfectedcells.Inourexperiencesomecelltypeseitherexpresscellsurface
markerspoorly,orcan′tproperlytrafficthemtotheplasmamembrane,resultinginaninabilitytodetecttransfectedcells.Likewise,notallcells
toleratethemembraneboundformofGFP.Selectionofthemostappropriatereportershouldbemadebyassessingtransfectionefficiencyofthe
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reporteraswellastherelativeintensityofexpressioncomparedtountransfectedcontrols.Ideally,heterologousexpressionofthesemolecules
willbewelltoleratedandthisissuggestivethatthemarkershavelittletonoeffectoncellcycledistribution.
Onelimitationofthesetypesofflowcytometryapproachesisthattheyareonlyabletoestablishrelativeabundancesofcellcyclephases
comparedtooneanother.Forthisreasonitisambiguousifexpressionofp27inFigure3trulyinducesanarrestinG1andG2/M,orifitjustslows
progressionthroughthesephasesrelativetoS-phase.Thesepossibilitiescanbeinvestigatedfurtherbytreatingaparallelsampleofcellswitha
mitoticinhibitorsuchasnocodazole,orG1/Sinhibitorlikeaphidicolin.SincethesedrugscreateadominantarrestinM-phaseorearlyS-phase
respectively,slowlyproliferatingcellswillaccumulateatthedruginducedarrestpoint.ForexamplecellsarrestedinG1becauseofpRB
expressionwillremaininG1despitenocodazoletreatmentwhilecontrolcellswillaccumulateinM-phase13.
Takentogether,theseexperimentalapproachesofferaflexiblemethodologythatcanbeappliedtoawiderangeofmammaliancellcycle
researchquestions.Theycanreadilydetectalterationsincellcycleprogressionandquantifydifferenceswhencomparedwithcontrols.
Disclosures
Theauthorshavenothingtodisclose.
Acknowledgements
TheauthorswishtothanktheCanadianInstitutesofHealthResearch(CIHR)andtheCanadianCancerSocietyforfundingcellcycleresearchin
theirlaboratory.MJCisarecipientofanMD/PhDfellowshipfromtheCIHR.MJCandMAaremembersoftheCaRTTtrainingprogram.
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