THAWINGCELLS

RemoveavialofPassage2PrimaryMouseEmbryoFibroblasts(PMEF"S),ataconcentrationof3x106fromliquidnitrogenandthawquicklyat37oC.

Addthe1mlofcellcontainingmediato9mlofwarmedPMEFmediaina15mlfalcontubeandspinonsettingoneforfiveminutes.ThisprocedureremovestheDMSO.

AspiratemediaoffthecellpelletandresUSPendgentlyin1mlofmediausinga1mlPipette.

Addtheresuspendedcellstoamedium(260ml)flaskcontaining10mlofPMEFmedia.

Incubateat37oCfortwodaysoruntilconfluent

IRRADIATINGCELLS

Oncecellsareconfluenttightenthelidoftheflaskandtakeuptothegammasourceintheanimalhouseonthethirdfloor(PMCI)

Irradiatetheflaskfor38minutes.(3000radsrequired.1gray=1.2718min,1gray=100rads).

Onceirradiated,wipeflaskwith70%ethanolandreturntoincubatoruntilreadytotrypsinise.

TRYPSINISINGCELLS

AspiratethePMEFmediafromtheflaskandrinsethecellswith10mlofwarmedPBS,aspiratingthePBSoff.

Add2mlofwarmed0.05%trypsin/EDTA,coveringthePMEFlayer.

Placetheflaskonthewarmingtrayfor2minutes,thengentlytapthesidesoftheflasktodislodgethecells.

Inactivatethetrypsinbyadding2mlofmedia.Gentlypipettethecellsupanddowntodisperseanyclumpsandobtainasinglecellsuspension.

Transferthe4mlofcellstoa15mlfalcontubeandcountthenumberofcells.

Spinthecellsononefor5minandresuspendataconcentrationof1x106cells/mlinmediacontaining10%DMSO.

Aliquot1mlintocyrotubesandfreezeinthe-70oCfreezerinaMrFrostyovernight.OncefrozentransferthevialstoboxD2inthe-70freezer.