细胞周期同步诱导方法.doc

细胞周期同步诱导方法.doc

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peteadam@

2013/1/10

DIAMONDSDeliverable1-D1.1.3

State-of-the-artinhumancellsynchronization.

DIAMONDSPARTNER:

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:

removeThymidinebywashingwith1xPBS;releasecellsbyaddingfreshDMEM（10%FCS,1%Pen-Strep,1%Glutamine）

ÞcellsprogresssynchronouslythroughG2-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:

removeThymidinebywashingwith1xPBS;

addfreshDMEM（10%FCS,1%Pen-Strep,1%Glutamine）for3htoreleasecells

-afterreleasingofthecellsadd100ng/mlNocodazoletotheMediafor

12h（mitoticblock）

-removeNocodazolebywashingwith1xPBSandaddfreshDMEM（10%FCS,1%Pen-Strep,1%Glutamine）toreleasecells

ÞcellsprogresssynchronouslythroughG1-andS-phase

FigureB. Cellswerearrestedinmitosisbyblockingfirstinthymidinefollowedbyreleaseandthenblockinginnocodazole.Afterreleasefromthenocodazoleblock,mostofthecells（>75%）dividedsynchronouslywithin2 hofreleasefromthearrest,enteredSphaseby10-12hafterrelease,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%Serum

Cserumstarvation

FigureC. Humandiploidfibroblasts（Tig3）werearrestedinG1-phasebyserumstarvation.Afterreleasefrom

G1block,CellsenteredSphaseby10-12hafterrelease,andcompletedthenextsynchronousmitosisby30-48h.

CellsynchronywasmonitoredbyflowcytometryofBrdUandpropidiumiodide-stainedcells.

4

D1.1.3:

Optimisedsynchronisationprotocolforyeasts,Arabidopsis,humancells