用DAPI和Hoechst33342染色法检测DNA的流式细胞方法探讨

#技术方法#用DAPI和Hoechst33342染色法检测DNA的流式细胞方法探讨刘锡娟v*,丁慧荣*,张宏(,,,100142)[]目的:DAPIHoechst33342DNA方法:DAPIHochestPI3HT-29,G0/G1SG2/MDNA,3DNA结果:,(CV)2.4;DAPIHoechst33342(chickenerythrocytenucle,iCEN)4,2341234,1CV2.4DAPIHoechst33342(calfthymocytenuclei,CTN)2,G2/G11.97,G0/G1CV2.4DAPIHoechst33342PI3HT-29,CV3.403.024.42,G0/G160.86%60.22%60.81%,S28.85%29.70%29.82%,G2/M10.29%9.09%9.37%,3结论:DAPIHoechst33342,DNA[];;DNA;[]R446-39[]A[]1671-167X(2010)04-0480-05do:i10.3969/.jissn.1671-167X.2010.04.028AmethodologystudyonflowcytometricanalysisofcellDNAstainedwithDAPIandHoechst33342LIUX-ijuanv*,DINGHu-irong*,ZHANGHong[KeyLaboratoryofCarcinogenesisandTranslationalResearch(MinistryofEducation),Centralab,PekingUniversitySchoolofOncology,BeijingCancerHospital&Institute,Beijing100142,China]ABSTRACTObjective:TodiscussasimplemethodforflowcytometricanalysisofcellDNAstainedwithDAPIandHoechst33342.Methods:HT29cellsstainedwithDAPI,Hoechst33342orPIweremeasuredbyBDFACSAriaandthepercentagesofcellsinG0/G1,SandG2/Mphaseswiththreestainingme-thods,thentheresultswereanalyzedandcompared.BeforemeasurementwemonitoredthequalityofDNAanalysisofflowcytometerthroughUVbeadsQCexperimentandanalyzedthestandardchickenerythrocytenuclei(CEN)andcalfthymocytenuclei(CTN)stainedwithDAPIandHoechst33342.Re-sults:CVvalueofUVpeakwas2.4afterQCexperiments.Therewere4peaksonCENhistogramsandtheratiosofpeakchannelmeanofG2/G1,G3/G1,andG4/G1wereabout2,3,and4respectively.BothCVvaluesofthefirstpeakwere2.4.Therewere2peaksonCTNhistogramsandtheratioofpeakchannelmeanofG2/G1was1.97,andCVvalueofG0/G12.4.ThecompletecellcycleofHT29cellsstainedwithDAPI,Hoechst33342orPIwasshowedentirely,CVvalueswere3.40,3.02and4.42,re-spectively,andthepercentagesofcellsinG0/G1were60.86%,60.22%and60.81%,respectively,inS,28.85%,29.70%and29.82%,respectively,andinG2/M,10.29%,9.09%and9.37%,re-spectively.Theresultsbythethreemethodsshowednodifference.Conclusion:ThismethodformeasurementofcellularDNAcontentisasimpleandefficientapproachtodeterminingcellcycleandcanbethefirstchoicewhenusingflowcytometerwith355nmUV.KEYWORDSStainingandlabeling;Flowcytometry;DNA;Cellcycle:(09-07)ThejointapplicationofLCMandSELDI-TOF-MS,2-dimensionalelectro-phoresistechnologyplatforms(09-07)vCorrespondingauthor.se-mai,[e-mail]*Theseauthorscontributedequallytothiswork,,[1]DNA,#480#()JOURNALOFPEKINGUNIVERSITY(HEALTHSCIENCES)Vo.l42No.4Aug.2010DNA,G0/G1%S%G2/M%,,[2](propidiumiodide,PI)DNA[3-4],,,488nm,610~620nm,RNase,RNADNADAPIHoechst33342DNA,DNAA-T,,355nm,400~500nmDAPIDNA,20,DNA,RNA,DNA[5],RNase,DAPIHoe-chst33342DNA,,,,DAPIHoechst33342DNA,70,DAPIDNA,DNA[4,6],,DNA,()90,[7-10],,BDFACSAria355nmDAPIHoechst33342HT29,DNA,DAPIHoechst33342,DNA,PI,11.1:BDFACSAria(qualitycontro,lQC)[UVbeadsDNAQCParticles(BectonDickinson)],OLYMPUSHT290.25%()(Gibico)1640(Gibico)FBS(Gibico)DAPIHoechst33342:,1g/L,,,4e;1@PBS1.2HT29,PBS2,1000r/min5min,500LLPBS,75%(),4e,1000r/min,,HT293,106/mL,1mLDAPI,5mg/L,30min,400;1mLHoechst33342,5mg/L,30min,400;RNase,1mLPI,50mg/L,30min,4001.3DNADNADNA,QCDNAQC,(chickenerythrocytenucle,iCEN)(calfthymocytenuclei,CTN)[1]1.3.1(CV)Bec-tonDickinsonQC,CV1.3.2DAPIHoechst33342CENCEN,40LL1mLDAPIHoechst33342,,10min,GlobalWorksheetFSC/SSCUV2-A/UV-WUV2-AFSC/SSCCEN,(R1),UV2-AR1,G0/G125,200001.3.3(doubletdiscriminationmod-ule,DDM)DAPIHoechst33342CTN,CTN,40LL1mLDAPIHoechst33342,,10min,Glo-balWorksheetFSC/SSCUV2-A/UV-W#481#,DAPIHoechst33342DNAUV2-AFSC/SSCCTN,UV2-A/UV-WUV2-AP1,G0/G150,200001.41.2DAPIHoechst33342HT29GlobalWorksheetFSC/SSCUV2-A/UV-WUV2-AFSC/SSC,(R1),UV2-AR1,G0/G150,200001.5DivaFSC2.0,ModFitLT,ModFitLTVeritySoftwareHouse,,DNA,DNAG0/G1CV22.1QC,,,QCCV(CV=2.4,1)2.2DNACEN,,DAPIHoechst333422;CTN,G0/G1,SG2/M,DAPIHoechst3334232.3HT29HT29DAPIHoechst33342PI,ModFitLT,G0/G1SG2/M,G0/G1CV3.403.024.42(4);G0/G160.86%60.22%60.81%,S28.85%29.70%29.82%,G2/M10.29%9.09%9.37%3,DNA3,,730,,,DNAG0/G1SG2/M,()G0G1G2MG1G1;,,,PIPE,FITC,;DAPIHoechst33342TexasRed,,PE,,[11],DAPIHoechstPI,RNase,,,,,DAPIPI,[12-13],,DAPIHoechst33342,,,,,DAPIHoechst,,350%~70%,,,DAPIHoechst[5-6],,,DAPI,24h,,,DAPI,,,,#482#()JOURNALOFPEKINGUNIVERSITY(HEALTHSCIENCES)Vo.l42No.4Aug.2010DNA,,DNA,,[1],,,(),G0/G1CVDNA,QC,UVbeads,CV2.5,,,G0/G1CV,,[1],DNA,DNA,,DDMCENCTN,,,CENDAPIHoechst33342,;CTN,,G0/G1,SG2/MCTNCEN,1#483#,DAPIHoechst33342DNACV,,,DDMDAPIHoechstPIHT-29,,3DNA,,DAPIHoechst33342PI,DNA,DNAPIDNA,(488nm),,,,,,DAPIHoechst33342,PBS,,,DNA,,DNA[1].[M].:,2008:471.[2].[M].:,2005:58.[3]NunezR.DNAmeasurementandcellcycleanalysisbyflowcy-tometry[J].CurrIssuesMolBio,l2001,3(3):67-70.[4]ChenXY,YangHX,QuSF,eta.lInvolvementofp38andc-JunN-terminalproteinkinaseincardiotoxinIII-inducedapoptosisofK562cells[J].BiolPharmBul,l2009,32(4):583-588.[5]KapuscinskiJ.DAPI:aDNA-specificfluorescentprobe[J].Bio-techHistochem,1995,70(5):220-223.[6]KrishanA,DandekarPD.DAPIfluorescenceinnucleiisolatedfromtumors[J].JHistochemCytochem,2005,53(8):1033-1036.[7]O.BrienMC,GuptaRK,LeeSY,eta.lUseofamultiparametricpaneltotargetsubpopulationsinaheterogeneoussolidtumormodelforimprovedanalyticalaccuracy[J].Cytometry,1995,21(1):76-83.[8