FreezingCells:1.Trypsinizecellsandharvestinthenormalway.2.Counta200ulaliquotanddeterminethetotalcellnumber.Fromthis,calculatethevolumeofmediarequiredtogiveafinalfreezingdensityof3.0x107cells/ml.3.Collectthecellsbycentrifugation@1,000rpmfor7minutes.4.AspirateoffthesupernatantandresUSPendthepelletin1/2thevolumecalculatedinStep2above.Usemediaappropriateforthecellsbeingfrozen(i.e.,M15forEScellsor7%FCS,1%GPSforSTO"s).5.Dilutethecellsuspension1:1with2XFreezingMedia(60%DMEM,20%FCS,20%DMSO;freshlyprepared).Addthemediadropwise,mixingwellaftereachaddition.6.Asepticallyaliquotthesuspensionintosterilefreezingvials,labeleachvialwiththedateandcelltype/clonenumber,andplacethevialsintoastyrofoamcontainer.7.Freezethecellsovernight@-70oC,thentransfertothe-135oCfreezer.ThawingOutCells:1.Removevialoffrozencellsfromthe-135oCfreezerandtransferto37oCwaterbathtothaw(thawinggenerallytakesonly1-2minutes).2.Transferthecellsuspensiontoasterile15mltube.Addappropriatemediadropwise,shakingthetubewellaftereachaddition."Topup"thetubewithadditionalmedia.3.Collectthecellsbycentrifugation@1,000rpmfor7minutes.4.Aspirateoffthesupernatantandresuspendthecellpelletin12mlofmedia.Plateoutthecellsona10cmplate(useagelledplateifplatingSTO"s;usea10cmfeederplateifplatingEScells).FromtheLaboratoryofDr.AllanBradleyBaylorCollegeofMedicine,Houston,Texas

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