ThawingCells IrrADIatingCells TrypsinisingCells- RemoveavialofPassage2PrimaryMouseEmbryoFibroblasts(PMEF"S),ataconcentrationof3x106fromliquidnitrogenandthawquicklyat37oC. - Addthe1mlofcellcontainingmediato9mlofPMEFmediaina15mlfalcontubeandspinonsettingoneforfiveminutes.ThisprocedureremovestheDMSO. - AspiratemediaoffthecellpelletandresUSPendgentlyin1mlofmediausinga1mlPipette. - Addtheresuspendedcellstoamedium(260ml)flaskcontaining10mlofPMEFmedia. - Incubateat37oCfortwodaysoruntilconfluent.Thensplit1:4andgrowuntilconfluent. - Oncecellsareconfluenttightenthelidoftheflasksandtakeuptothegammasourceintheanimalhouseonthethirdfloor(PMCI),ifyouhaveanumberofflasksyoucantrypsinisealltheflasksandplacethecellsuspensionsinafalcontubeandirradiatethat. - Irradiatetheflaskfor41minutes.(3000radsrequired.1gray=1.376min(Oct.98),1gray=100rads). - Onceirradiated,wipeflaskwith70%ethanolandreturntoincubatoruntilreadytotrypsinise. - AspiratethePMEFmediafromtheflaskandrinsethecellswith10mlofwarmedPBS,aspiratingthePBSoff. - Add2mlofwarmed0.05%trypsin/EDTA,coveringthePMEFlayer. - Placetheflaskonthewarmingtrayfor2minutes,thengentlytapthesidesoftheflasktodislodgethecells. - Inactivatethetrypsinbyadding2mlofmedia.Gentlypipettethecellsupanddowntodisperseanyclumpsandobtainasinglecellsuspension. - Transferthe4mlofcellstoa15mlfalcontubeandcountthenumberofcells. - Spinthecellsononefor5minandresuspendataconcentrationof1x106cells/mlinmediacontaining10%DMSO. - Aliquot1mlintocyrotubesandfreezeinthe-70oCfreezerinaMrFrostyovernight.OncefrozentransferthevialstoboxD2inthe-70freezer.
