细胞周期同步诱导方法VIP免费

peteadam@yeah.net2013/1/10DIAMONDSDeliverable1-D1.1.3State-of-the-artinhumancellsynchronization.DIAMONDSPARTNER:KristianHelin,BiotechResearchandInnovationCentre(BRIC),UniversityofCopenhagen,DenmarkSynchronizationprotocolsforhumancellsI.)DoubleThymidineblock(earlyS-phaseblock)-at25-30%confluencyofHeLacellculturewashtwicewith1xPBSandaddDMEM(10%FCS,1%Pen-Strep,1%Glutamine)+2mMThymidinefor18h(firstblock)-afterfirstThymidineblock:removeThymidinebywashingwith1xPBS;addfreshDMEM(10%FCS,1%Pen-Strep,1%Glutamine)for9htoreleasecells-afterreleasing:addDMEM(10%FCS,1%Pen-Strep,1%Glutamine)+2mMThymidinefor17h(secondblock)-aftersecondblock:removeThymidinebywashingwith1xPBS;releasecellsbyaddingfreshDMEM(10%FCS,1%Pen-Strep,1%Glutamine)cellsprogresssynchronouslythroughG2-andmitoticphaseD1.1.3:Optimisedsynchronisationprotocolforyeasts,Arabidopsis,humancells1peteadam@yeah.net2013/1/10FigureA.SynchronyofHeLacells.(A)CellswerearrestedatthebeginningofSphasebyusingadoublethymidineblock,andcellsynchronywasmonitoredbyflowcytometryofpropidiumiodide-stainedcells.Flowcytometrydatawerecollectedforeachofthethreeindependentdoublethymidineblocksperformedinthisstudy;dataareshownonlyfortheseconddoublethymidinearrest(Thy-Thy2),althoughequivalentsynchronywasobtainedineachofthethreeexperiments.Thenumberofcells(arbitraryunits)isplottedagainstDNAcontentfortimepointsat4-hintervalsfor44h;anarrowindicatesthetimeofmitosis,asestimatedfromtheflowcytometrydata.Uponreleasefromthethymidineblock,>95%ofthecellsprogressedintoSphase(0-4h),enteredG2phase(5-6h),underwentasynchronousmitosisat7-8h,andreenteredSphaseaftercompletingonefullcellcycleat14-16h.Typicallytwotothreeadditionalsynchronouscellcycleswereobtained.MCBVol.13,Issue6,1977-2000,June2002,MichaelL.Whitfieldetal.II.)Thymidine-Nocodazoleblock(mitoticblock)-at40%confluencyofHeLacellcultureaddDMEM(10%FCS,1%Pen-Strep,1%Glutamine)+2mMThymidinefor24h(S-phaseblock)-afterThymidineblock:removeThymidinebywashingwith1xPBS;addfreshDMEM(10%FCS,1%Pen-Strep,1%Glutamine)for3htoreleasecells-afterreleasingofthecellsadd100ng/mlNocodazoletotheMediafor12h(mitoticblock)-removeNocodazolebywashingwith1xPBSandaddfreshDMEM(10%FCS,1%Pen-Strep,1%Glutamine)toreleasecellscellsprogresssynchronouslythroughG1-andS-phaseFigureB.Cellswerearrestedinmitosisbyblockingfirstinthymidinefollowedbyreleaseandthenblockinginnocodazole.Afterreleasefromthenocodazoleblock,mostofthecells(>75%)dividedsynchronouslywithin2hofreleasefromthearrest,D1.1.3:Optimisedsynchronisationprotocolforyeasts,Arabidopsis,humancells2peteadam@yeah.net2013/1/10enteredSphaseby10-12hafterrelease,andcompletedthenextsynchronousmitosisby18-20h,ultimatelycompletingtwofullcellcycles.MCBVol.13,Issue6,1977-2000,June2002,MichaelL.Whitfieldetal.III.)Serumstarvation(G0/G1block)-at30-40%cellconfluencywashtwicewith1xPBSandaddDMEM(1%Pen-Strep,1%Glutamine)w/oSerum-after72hrestimulationwith10-15%SerumCserumstarvationFigureC.Humandiploidfibroblasts(Tig3)werearrestedinG1-phasebyserumstarvation.AfterreleasefromG1block,CellsenteredSphaseby10-12hafterrelease,andcompletedthenextsynchronousmitosisby30-48h.D1.1.3:Optimisedsynchronisationprotocolforyeasts,Arabidopsis,humancells30200400600800100002004006008001000020040060080010000200400600800100002004006008001000020040060080010000h6h12h24h30h48hpeteadam@yeah.net2013/1/10CellsynchronywasmonitoredbyflowcytometryofBrdUandpropidiumiodide-stainedcells.D1.1.3:Optimisedsynchronisationprotocolforyeasts,Arabidopsis,humancells4