细胞周期同步诱导方法文档格式.doc
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细胞周期同步诱导方法文档格式.doc
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KristianHelin,
BiotechResearchandInnovationCentre(BRIC),UniversityofCopenhagen,Denmark
Synchronizationprotocolsforhumancells
I.)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:
releasecellsbyaddingfreshDMEM(10%FCS,1%Pen-Strep,1%Glutamine)
Þ
cellsprogresssynchronouslythroughG2-andmitoticphase
FigureA.
SynchronyofHeLacells.(A)CellswerearrestedatthebeginningofSphasebyusingadoublethymidineblock,andcellsynchronywasmonitoredbyflowcytometryofpropidiumiodide-stainedcells.Flowcytometrydatawerecollectedforeachofthethreeindependentdoublethymidineblocksperformedinthisstudy;
dataareshownonlyfortheseconddoublethymidinearrest(Thy-Thy2),althoughequivalentsynchronywasobtainedineachofthethreeexperiments.Thenumberofcells(arbitraryunits)isplottedagainstDNAcontentfortimepointsat4-hintervalsfor44
h;
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:
addfreshDMEM(10%FCS,1%Pen-Strep,1%Glutamine)for3htoreleasecells
-afterreleasingofthecellsadd100ng/mlNocodazoletotheMediafor
12h(mitoticblock)
-removeNocodazolebywashingwith1xPBSandaddfreshDMEM(10%FCS,1%Pen-Strep,1%Glutamine)toreleasecells
Þ
cellsprogresssynchronouslythroughG1-andS-phase
FigureB.
Cellswerearrestedinmitosisbyblockingfirstinthymidinefollowedbyreleaseandthenblockinginnocodazole.Afterreleasefromthenocodazoleblock,mostofthecells(>
75%)dividedsynchronouslywithin2
hofreleasefromthearrest,enteredSphaseby10-12hafterrelease,andcompletedthenextsynchronousmitosisby18-20h,ultimatelycompletingtwofullcellcycles.
III.)Serumstarvation(G0/G1block)
-at30-40%cellconfluencywashtwicewith1xPBSandaddDMEM(1%Pen-Strep,1%Glutamine)w/oSerum
-after72hrestimulationwith10-15%Serum
Cserumstarvation
FigureC.
Humandiploidfibroblasts(Tig3)werearrestedinG1-phasebyserumstarvation.Afterreleasefrom
G1block,CellsenteredSphaseby10-12hafterrelease,andcompletedthenextsynchronousmitosisby30-48h.
CellsynchronywasmonitoredbyflowcytometryofBrdUandpropidiumiodide-stainedcells.
4
D1.1.3:
Optimisedsynchronisationprotocolforyeasts,Arabidopsis,humancells
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- 细胞周期 同步 诱导 方法