u2os drgfp hrWord文件下载.docx
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vefirstthawedout.Swirlthetubein37°
Cwaterbathuntilmelted.
4.Underhood,changethepipetterto“slow”speed.Remove2mLofthemediafromthefalcontubeandslowlyaddthemediatothecryotube,mixingthecellswithmedia.Slowlyaddthemixedmediawithcellsbacktothe15mLfalcontube,slowlypipettingupanddowntomix.
5.Spincellsinthetable-topcentrifuge3minutesat1000rpm.Iftherearefewcells,spininswingingbuckettopelletcellsatthebottomofthetube.
6.Aspirateoffsupernatant.Add2mLoffreshmediaandslowlypipetteupanddowntomix.Thiswillbreakupanyclumps.Ideallyyouwantsinglecells.
7.Addcellsandmediato10cmplate.Mixbyswirlingtoensureevencoverageonthebottom.
8.Lookatcellsat4Xor10Xscopetomakesurecellshavebrokenup.
9.Moveplatetoincubator.
10.Thenextday:
checkcells-ifconfluent,split1:
5to2x15cmplates
11.U2OScellsgenerallyneedtobesplitevery2-3daysat1:
3to1:
5.
Day0:
splitcellsfromaconfluent15cmplate1:
3to2x15cm:
1.Aspirateoffmedia.
2.washwith10mLPBS;
aspirateoffPBS
3.add3mLtrypsin(warmedto37°
C)toeachplate.
4.incubateat37°
Cfor3-5minutesoruntilcellsbecomedetached)
5.whilecellsareincubating,preparenewplatestosplittobyadding20mLpre-warmedmedia
6.add6mLmediatoeachplatewithtrypsinizedcells
7.resuspendcellstomakesingle-cellsuspension
8.add3mLofcellsuspensionto2new15cmplateswith20mLofprewarmedmedia.
9.placeinincubators
Day1:
DNATransfectionsPreparecells:
U20SDRGFPHRassay
J.LaRocque,adaptedfromJasinlab
06/01/2011
1.trypsinizecellsasonDay0
2.poolcellsinto50mLfalcontube
3.dilute100uLcellsuspensioninto500uLPBS
4.usedilutedcellsuspensiontocountcellsusinghemocytometer
5.resuspendcellsinOPTI-MEMorPBSto7.7x106cells/mL
PrepareDNA:
add25pigofplasmidintoeachcuvette:
A:
CAGGS(noDSBcontrol)
B:
CBAS(inducesDSB)
C:
NZE-GFP(measurestransfectionefficiency)
Electroporatecells:
1.add0.650mLofresuspendedcellsintoeachcuvette(5x106cellseachtransfection)
2.setBio-RadGenePulsarIIelectroporator:
250V;
950|iF
3.Pulse,add0.5mLmediatocuvette,thentransferto10cmplatewith8mLmedia
4.Putinincubatorfor48hours
Day3:
(48hrsposttf):
FACSanalyses
Preparecells:
1.trypsinizecellsaspreviously,usingonly1mLtrypsinfor10cmplates
2.add2mLmediaandresususpendcellsintosingle-cellsuspension
3.transfer1mLofcellsuspensionintoFACstubes(Falcon#352008).FACsfrommedia;
cannotfixcellsduetoGFPleakagefromcells,somustFACswithinanhour.
FACScells:
atapprox100-400cells/second.
FACScansettings:
Detectorvoltageampgainmode
FSC E-1 4.82 LOG
SSC 228 1.0 LOG
FL1 461 LIN
FL2 549 LIN
FL3NA
FL210%FL1
Reeagentsandmaterials:
U2OSmedia:
500mLDMEM-HG
50mLFetalBovineSerum(GeminiBio-Products;
cat#100-106althoughanyFBSshouldbefine)
5.5mLPen-StrepSolution100X(GeminiBio-Products)
J.LaRocque,adaptedfromJasinlab06/01/20111XPBS(noMgorCa)
Trypsin0.2%;
ImMEDTAinPBSw/oMgorCaw/ophenolred
Electroporationcuvettes(Bio-Rad0.4cm;
cat#1652088)
15cmplates(Nunc#12-565-100)
10cmplates(BDFalcon#08-772E)
15mLfalcontube
50mLfalcontube
5mLtubesforFACS(falcon#352008)
hemocytometer
OPTI-MEM(invitrogen)-CanalsousePBSisyoudon'
thaveOPTI-MEMinBioRadGenePulserII