细胞周期的流式细胞伩检测实验方法（PI，Brdu)
ANALYSISOFCELLCYCLEMiriamCapriandDanielaBarbieriDept.BiomedicalSciences,Sect.GeneralPathology,ViaCampi,287,UniversityofModena,41100Modena,Italy 1.INTRODUCTION A)Bromodeoxyuridine/PropidiumIodideTheclassicalmethodfortheanalysisofcellcycledistributionistheflowcytometricmeasurementofDNAcontentwhichcansimultaneouslydeterminetheincorporationofBromodeoxyuridine(BrdU).TheprocedurerequiresthatDNAispartiallydenaturedtoexposeincorporatedBrdUtoaspecificantibody.DenaturationisnecessarybecauseantibodiesdevelopedsofarbindonlytoBrdUinsingle-strandDNA.TheremainingundenaturedDNAisthenstainedwithPropidiumIodide(PI).Greenfluorescencefromthefluorescein-conjugatedantibodyisameasureofBrdUincorporation.RedfluorescencefromthePIisameasureofDNA.Theprotocoldescribedhereuseshigh-molarityHClforthedenaturationofDNA.FurThermore,thismethodmaybeutilizedeitherforunfixedorforfixedcellsinsUSPension. B)Cyclins/PropidiumIodideCyclinsarekeycomponentsofthecellcycleprogressionmachinery.Inparticular,theexpressionofcyclinsD,E,AandB1providesnewcellcyclelandmarksthatcanbeusedtosuBDividecellcycleintoseveraldistinctsubcompartments.Inthisprocedurecyclinsexpressionisdetectableusingspecificmonoclonalantibodies(mAbs),andisanalysedinrespecttoDNAcontent. Generally,thepeakofexpressionofcyclinD1canbedetectedinearlyG1,thepeakofcyclinEistypicalofG1/Stransition,thepeakofcyclinAcanbedetectedduringG2/MphasesandcyclinB1istypicaloflateG2/M.Usingthismethod,comparedtotheabovementionedprotocol,itispossIBLetodistinguishG0fromG1andG2fromMphases.However,itisnecessarytokeepinmindthatnotallcelltypesbehaveinthesamemanner(forexample,cyclinD1isdetectablenotonlyinG0/G1butalsoinG2/M,evenifinaveryfewcelltypes). C)TUNEL/PropidiumIodideOneofthemostusedprotocolforthedeterminationofapoptosisinthedifferentphasesofcellcycleistheenzymaticinsitulabelingofapoptosis-inducedDNAstrandbreaks(TUNEL).Terminaldeoxynucleotidyltransferase(TdT)havebeenusedfortheincorporationoffluorescein-labelednucleotidestoDNAstrandsbreaksinsitu.DNAcontentisrevealedbyredfluorescencefromPI.Inordertohavemoredetails,seetheChaptersrelatedtoTUNELtechnique. D)F-Actin/PropidiumIodideTheanalysisofapoptoticcellsandestimationoftheircellcyclespecificityisalsopossibleusingarecentmethod.ThisisbasedonidentificationofapoptoticcellswhichhavemodifiedtheircytoskletonandtheirDNAcontent.Inspecific,paraformaldehyde(PFA)fixationfollowedbystainingofF-actinwithfluorescein-conjugatedphalloidinandofDNAwithPI,areused.Furthermore,thisproceduremaybeutilizedalsoforadherentcells. 2.PROTOCOLS 1.Cells(1x106/mL)areincubatedwithBrdU10mMatfinalconcentration,for30minat37°Cincontrolledatmosphere.2.Washtwiceat500gfor1minusingthewashingbuffer.3.Resuspendin0.5mLofwashingbufferand0.5mLofHCl4M.4.Mixaccuratelyandincubatefor30minatroomtemperature.5.Washonceasinstep2.6.Resuspendin1mLofBoraxbuffer.7.Asinstep5.8.Resuspendin200mLofwashingbufferandlabelwith5mLofmAbant-BrdU.9.Incubatefor1hourat4°Cinthedark.10.Asinstep5.11.Resuspendin200mLofwashingbufferandlabelwith4mLofgoat-anti-mouseFITC-conjugatedantibody.12.Incubatefor30minat4°Cinthedark.13.Asinstep5.14.Resuspendin200mLofwashingbufferand200mLofPIbuffer.15.Incubatefor15-30minat4°Cinthedark.16.Analysewithflowcytometerequippedwitha488nmargonlaser. A.3.COMMENTARY A.3.1BackgroundinformationInthisprocedurefixedcellsby4%PFAinPhosphateBufferSaline(PBS)canbeutilized.InthiscasetowashcellsonceinPBSbeforetostartatstep1isnecessary. Moreover,bothdirectandindirectimmunofluorescencecanbeused.TheBrdUincorporationismoreevidentusingtheindirectmethod. B)Cyclins/PIprotocol B.2.1MaterialsPFA(A2),TritonX-100(A2),PBS,mouseserum(A2),mAbsanti-cyclins(A2),goat-anti-mouse-FITC(A2),PI(A2),GM+EDTAbuffer(A1). InthecaseofE,AandB1cyclins,cells(2x106/mL)arefixedby70%ethanol: a)putallthereagentsinice;b)countandcentrifugecellsat375gfor5min;c)resuspendaccuratelythepelletin1mLGM+EDTAbuffer;d)addgently3mLof96%ethanolvortexingsamplesandworkinginice;e)storethefixedsamplesat4°C. InthecaseofDcyclins,cellsarefixedby1%methanol-freeformaldehyde: a)putallthereagentsinice;b)countandcentrifugecellsat375gfor5min.;c)resuspendgentlythepelletin1mLof1%formaldheydeinPBSfor15mininice;d)washinice-coldPBSasinstepb;e)add1mLof70%ethanol;f)storethefixedsamplesat4°C. STAINING 1.Washwithice-coldPBS. 2.Resuspendthepelletin1mLof0.25%TritonX-100inPBS,vortexingsamplesandworkinginice(for5mininthecaseofD,AandB1cyclins,for10-20mininthecaseofEcyclin). 3.Washinice-coldPBSasinstepb. 4.Incubatecellswith150mLofmAbanti-cyclin(diluitedattheconcentrationof2.5mg/mLinPBS+1%ofnormalgoatserum)overnightat4°C. 5.Asinstep3. 6.Incubatefor1hourwith150mLsecondarymAbgoat-anti-mouse-FITC(1.3mg/mL)diluited1:50inPBS+1%normalgoatserum. 7.Asinstep3. 8.Incubatefor4hoursatroomtemperatureandinthedarkwith1mLof2.5mg/mLPIinPBS(+12.5mLof1mg/mLRNAsi)orincubateovernightat4°Cwith0.8mg/mLPIinPBS(+12.5mLof1mg/mLRNAsi). 9.Analysewithflowcytometerequippedwitha488nmargonlaser. B.3.COMMENTARY B.3.1BackgroundinformationThecriticalstepsinthemethodologyarecellfixation,permeABIlizationandtheconcentrationsofanti-cyclinmAbs.Formostcyclinsoptimalfixationis70%ethanol.Thistreatmentpreservescyclins,loweringthebackground,non-specificcellfluorescenceandresultinginanimprovedsignal-to-noiseratioofthecyclinspecificfluorescence.DetectionofDcyclin,however,requiresfixationinformaldehyde.Asfarasanti-cyclinmAbconcentrationisconcerned,2.5mg/mLisoptimalformostcells.Anyway,totestthebestconcentrationforeachexperimentalmodel,isrecommended. B.3.2AnticipatedresultsInthisprocedure,anegativecontrolsample,whichcontainsonlythesecondaryFITC-mAb,isnecessary. B.3.4Keyreferences 2.Faretta,M.,Bergamaschi,D.,RonzoniS.,D’Incalci,M.,Erba,E.1997.DiferencesincyclinB1expressionincellcycleblockedintheG2/Mphaseaftertreatmentwithanti-canceragent.Anewthreeparametricflowcytometryanalysis.ProceedingsoftheXIVNationalItalianMeetingofCytometry. 3.Gong,J.,Traganos,F.,Darzynkiewicz,Z.1993.SimultaneousanalysisofcellcyclekineticsattwodifferentDNAploidylevelsbasedonDNAcontentandcyclinbmeasurements.CancerRes.53:5096. 4.Gong,J.,Li,X.,Traganos,F.,Darzynkiewicz,Z.1994.ExpressionofG1andG2cyclinsmeasuredinindividualcellsbymultiparameterflowcytometry:anewtoolintheanalysisofthecellcycle.CellProlif.27:357. 5.Gong,J.,Traganos,F.,Darzynkiewicz,Z.1995.DiscriminationofG2andmitoticcellsbyflowcytometrybasedondifferentexpressionofcyclinsAandB1.Exp.CellRes.220:226. 6.Widrow,R.J.,Rabinovitch,P.S.,Cho,K.,Laird,C.H.1997.SeparationofcellsatdifferenttimeswithinG2andmitosisbycyclinB1flowcytometry.Cytometry27:250. C)TUNEL/PIprotocol C.2.1Materialsformaldehyde(A2),ethanol,reactionmixture(A1),TdTbuffer(A1),Bio-16-dUTP(A2),TdTenzyme(A2),stainingbuffer(A1),SSCbuffer(A1),BLOTTO(A2),Avidin-FITC(A2),TritonX-100(A2),PI(A2),DNAasebuffer(A1). C.3.COMMENTARY C.3.1BackgroundinformationThisprocedureiscomplexandnotalwaysgoodresultsareobtained.Thus,theuseofcommercialkitssuchasApoTagTM(Oncor,Gaithersburg,MD,USA)and\"InsitucelldeathdetectionKit\"(Boeringer-Mannheim,Germany),ishighlyrecommended. 2.Gorczyca,W.,Tuziak,T.,Kram,A.,Melamed,M.R.,Darzynkiewicz,Z.1994.Detectionofapoptosis-associatedDNAstrandbreaksinfine-needleaspirationbiopsiesbyinsituendlabelingoffragmentedDNA.Cytometry15:169. 3.Li,X.,Darzynkiewicz,Z.1995.LabellingDNAstrandbreakswithBrdUTP.Detectionofapoptosisandcellproliferation.CellProlif.28:571. D)F-Actin/PIprotocol D.2.1MaterialsPFA(A2),PBS,TritonX-100(A2),sodiumborohydride,FITC-phalloidin(A2),PI(A2). 1.Cellsarefixedin1mLof1%PFAfor30minonice.2.Washwith0.1%TritonX-100inPBS,andincubatewith0.1%sodiumborohydrideinPBS(pH8.0)for30min.3.Washat200gfor5min.4.Incubatewith20mLofFITC-phalloidin(0.01-10.0mg/mL)for1houratroomtemperature(orovernightat4°C).5.Asinstep3.6.Resuspendedin1mLofa5-50mg/mLPIinPBSandincubatefor30minat37°C.7.Analysedwithflowcytometerequippedwitha488nmargonlaser. D.3.COMMENTARY D.3.1BackgroundinformationUsingthisprotocol,theacquisitionandanalysisofthesamplesisparticularlyimportant.Apoptoticandnonapoptoticcellsaredistinguishedonthebasisofthegreenflourescenceandthesidescatter.ApoptoticcellshavehighsidescatterandlowFL-1(1).TheanalysisofDNAcontentisrelativetothedifferentregionsofapoptoticandnonapoptoticcells. ii)washat200gfor5min(continuetostep1). D.3.3TimeconsiderationsTheprotocolisrelativelysimpleandfast,inparticular2hoursandhalfarebasicallynecessary. D.3.4Keyreferences1.Endresen,P.C.,Prytz,P.S.,AarbalckeJ.1995.AnewflowcytometrymethodfordiscriminationofapoptoyiccellsanddetectionoftheircellcyclespecificitythroughstainingofF-ActinandDNA.Cytometry.20:162. Appendix1:Stocksolutions Appendix2:Reagents sc-594 sc-717,sc-753,sc-618 Appendix3:Equipment
Cellcycleandapoptosisareveryimportantfunctionalparameterstoassessthecellularmetabolism,physiologyandpathology.Severaltechniqueshavebeendevelopedtoquantitatetheseparametersutilizingthedifferentialstainingoffluorescentdyes.Wearedescribingfourdifferentflowcytometricmethods,twoforthediscriminationofcellcyclephases(AandB)andtwoforthesimultaneousassessmentofcellcycleandapoptosis(CandD).
A)BrdU/PIPROTOCOLA.2.1MaterialsBrdU(A2),washingbuffer(A1),HCl4M,Boraxbuffer(A1),anti-BrdUantibody(A2),goat-anti-mouse-FITCantibody(A2),PIbuffer(A1).
A.2.2.MethodologyA.3.2AnticipatedresultsItisrecommandedtoperformeachexperimentusinganegativeandapositivecontrolsample.Thenegativesample,inordertohaveacorrectsettingofinstrument,isassessedfollowingallthestepsexceptstep8.Thepositivesample,inordertomakesurethatthemethodworks,isassessedbyusingaproliferatingcellline(suchasU937,K562,MOLT4,etc.)followingallsteps.
A.3.3TimeconsiderationsTheprotocolissimplybutitrequireaquitelongtime.Indeed,forfewsamples,moreorless4hoursarerequired.Thedurationofthemethodisobviouslydependingonthenumberofsamples.A.3.4Keyreferences1.Dolbeare,F.,Gratzner,H:,Pallavicini,M.,Gray,J.W.1983.Proc.Natl.Accad.Sci.U.S.A.80:5573.C.2.2.Methodology1.Cells(5x105-1x106cell/sample)arewashedtwiceat260gfor5minusing2mLofPBS(pH7.2).
2.Fixby1mLof1%formaldehydeinPBS,onicefor15min.3.Washonceasinstep1.4.Resuspendin1mLofice-coldethanol70%(atthispointtostorethesamplesat-20°Cfor18hoursorovernightispossible).5.Asinstep3.6.Resuspendin50mL(foreachsample)ofthereactionmixture,whichispreparedduringthelastspindown.7.Incubatefor30minat37°Cwaterbath.8.Asinstep3.9.Resuspendin100mL(foreachsample)ofthereactionstainingbuffer,whichispreparedduringthelastspindown.10.Incubatefor30minatroomtemperatureinthedark.11.Asinstep3.12.CounterstainDNAwith5mg/mLofPIinPBS.13.Incubatefor15minat4°Cinthedark.14.Analysewithflowcytometerequippedwitha488nmargonlaser.C.3.2AnticipatedresultsToperformeachexperimentusingablank,anegativeandapositivecontrolsamples,isrecommended.Theblanksampleisassessedsubstitutingstep9asfollowing:add100mLofreactionstainingbufferpreparedwithoutAvidin-FITC.Thenegativesampleisassessedsubstitutingstep6asfollowing:add50mLofreactionstainingbufferpreparedwithoutTdTenzyme.Blankandnegativeareperformedinordertohaveacorrectsettingoftheinstrument.Thepositivesample,inordertomakesurethatthemethodworks,isassessedbydigestingwith75mLofDNAsebufferfor20minatroomtemperaturebeforestep6.
C.3.3TimeconsiderationsTheprotocolrequireaquitelongtime.Inparticular1hourandhalfbeforetheovernightincubationandacoupleofhoursafter.Obviously,utilizingcommercialkitsthedurationofmethodishighlyreduced.C.3.4Keyreferences1.Gorczyca,W.,Gong,J.,Darzynkiewicz,Z.1993.DetectionofDNAstrandbreaksinindividualapoptoticcellsbytheinsituterminaldeoxynucleotidyltransferaseandnicktranslationassays.CancerRes.52:1945.
Inthisprocedureadherentcellscanbeutilized.Inthiscaseitisnecessarytostartthemethodasfollowing:
i)addPFA2%directlytothecultureflasksfor30minonice.Avolumeequaltothatinthecultureflasksisadded,making1%thefinalPFAconcentration;Solution Preparation Storage A.Washingbuffer 0.5%Tween20inPBS 4°C A.Boraxbuffer 0.1MBorax(Sodiumtetraborate-10-hydrate) RT A.PIbuffer 3.4mMTrisodiumCitrate,9.65mMNaCl,PI20mg/ml,0.03%NonidetP-40inH2O 4°C B.GM+EDTAbuffer glucose1.1g/L,NaCl8g/L,KCl0.4g/L,Na2HPO4.2H2O0.2g/L,KH2PO40.15g/L,EDTA0.2g/L 4°C C.reactionmixture 50mLofsolutionwascomposedby:37.8mLofdeionizedwater+5mLofTdTbuffer(10X),+5mLofCoCl2(25mM),+2mLofBio-16-dUTP+0.2mLTdTenzyme 0°C C.TdTbuffer(10X) 1MNacacodylate(pH7.0),1mMdithiothreitol,0.5mg/mLserumalbumin 4°C C.stainingbuffer 100mLofsolutionwascomposedby:54.2mLofdeionizedwater+SSCbuffer(20X),+20mLofBLOTTO(25%)+0.7mLAvidin-FITC(160X),+0.1mLofTritonX-100 4°C C.Avidin-FITC160X 1mgAvidin-FITCin250mLPBS.Thendiluit1/10indeionizedwatertohave160Xstock 4°C C.SSCbuffer(20X) 0.3%sodiumcitrate,3MNaCl(pH7.0) RT C.DNAasebuffer 20ng/mLDNAasi,10mMTRIS-HCl(pH7.4),10mMNaCl,5mMMgCl2,0.1mMCaCl2,25mMKCl 0°C Avidin-FITCSigmaAldrich A2910 anti-BrdUantibodyBectonDickinson 347580 Bio-16-dUTP(50nmol/50mL)BoehringerMannheim 1093070 BLOTTO(drynon-fatmilk)Bio-Rad 170-6404 BoraxRiedel-Dehaen 31457 BrdUSigmaAldrich B5002 DNAasiBoehringerMannheim 776785 FITC-phalloidinSigmaAldrich P5282 formaldehydeBDH 10113 goat-anti-mouse-FITCantibodyBectonDickinson 349031 AntiAcyclinantibodySantaCruzBiotechnology sc-239,sc-596,sc-751 AntiB1cyclinantibodySantaCruzBiotechnology sc-245,sc-752,sc-595, AntiD1cyclinantibodySantaCruzBiotechnology sc-6281,sc-246,sc-450, AntiEcyclinantibodySantaCruzBiotechnology sc-247,sc-198,sc-481 mouseserumCaltag 10410 NonidetP-40SigmaAldrich N6507 paraformaldehydeSigmaAldrich P6148 PISigmaAldrich P4170 BovineserumalbuminSigmaAldrich B7276 TritonX-100SigmaAldrich T9284 TdTenzyme(25U/mL)BoehringerMannheim 220582 Tween20Merk-Schuchardt 822184 FlowCabinetTC60 Gelaire FlowCytometerFACScan BectonDickinson IncubatorCO2-AUTO-ZERO Heraeus MinifugeRF Heraeus PipetmanP20,P200,P1000 Gilson VortexVibrofixVF1Electronic Janke&Kunkel-IkaLabortechnik WaterBathD8 Haake
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