Cellarenormallypassagedevery2-3days,thisisimportanttoavoiddifferentiation.

Signsofdifferentiationare:-

i)coloniesaresurroundedbyflatteneddifferentiatedcells.

ii)largecolonieswithnecroticcentres,theseappearascellswithdefinedboundaries.

iii)coloniesappearasindividualcellsratherthanasasyncialmass.

iv)coloniesaremore"rounded"than"flat",theyalsohaveaclearlydefinedboundary,worsethanthis,theyhaveformedfreefloatingembryoidbodies.

Cellsarepassagedasfollowed:-

1.Allreagentsarewarmedto37

Medium

PBS

PBS/EGTA

TRYPSIN/EDTA

2.Removemedium

3.WashwithPBS(5ml/25cm2flask,10ml/80cm2flask),aspirate

4.WashwithPBS/EGTA,aspirate

5.Placeflaskon37warmingtrayforapprox.1minoruntilindividualcellscanbeseenincolonies.

6.Add0.5ml(25cm2),1ml(80cm2)TRYPSIN/EDTAandrockflaskbackwardsandforwardsuntilcoloniesfloatoff,thisshouldtake~1min.

7.Usinga1mlPipette,pipetteupanddown,(avoidmakingbubblesasthesekillthecells),forapprox.1min.CheckthatallcolonieshavebeendispersedandthatasinglecellsUSPensionhasbeenachieved.Don"tleavecellsinTYRPSIN/EDTAforlongerthan3minsasitisquitetoxic.

8.Neutralisetrypsinbyaddinganequalvolumeofmedium,mixbygentlepipetting.

9.Aspiratemediafromfeederflask,asthisisifferentmediatoEScellmedia.Seedfeeder*flaskswithanaliquotofcellsuspension,a1:10and1:20splitisappropriateforawellgrowingculturewithmedium-largecoloniesthatarenottouchingeachotherbutarereasonablyclosetogether.

*FeederflaskscontainMitomycinCtreated(seeprotocol)PrimaryMouseEmbryoFibroblasts(PMEFs)ataconcentrationof0.3x106/25cm2flask,1x106/80cm2flask,orMitomycinCtreatedSTOcellsataconcentrationof1.25x106/25cm2flask,4x106/80cm2flask.STOcellsaresmallerthanPMEF"s.(seecharts).

IfcellsaretobegrowninthepresenceofLIFonlyie.nofeederlayer,flasksorplatesmustbetreatedwith0.1%gelatininPBSat37foratleast1hr-2hrs.Thisisremovedbeforethemediumisadded.