FEEDERPREPARATIONBYGAMMAIRRADIATIONAllproceduresshouldbecarriedoutusingsteriletechniquesatalltimes.MediaforSTOs/SNL76/7cellsisDMEM(highglucose,nopyruvate),7%FBSand1XGPS.1.Gelatinizeplates,atroomtemperaturefor2hours.2.Aspiratemediaoffthe15-cmplates.3.Washcells1XwithPBS.AspirateoffthePBS.4.Add2.5mlofTrypsintoeachplate(15cm),coverthesurfaceentirely.5.Incubateplates@37C,for5min.6.Add5-6mloffeedermedia(DMEM,7%FBS,1XGPS)toeachplate.Thisinactivatesthetrypsin.7.HarvestcellsbypipetingupanddownthecellsUSPensionandtransfertosterile50mlcentrifugetube.Repeattheprocessandpoolallplatesinto1tube.Youcanpool5x15cmdishesinto1x50mlcentrifugetube.8.Countbeforespin,withtheCoulterCounter.9.Next,dispenseexactly6-7mlsofcellsuspensionpereach15mltubes;youwillhaveasaresult7x15mltubesperevery50mlcentrifugetube.10.Cellsarenowreadytobegammairradiated.YouneedtoadmiNISTer6,000rads.IfyouhavenotusetheIrradiator,youneedtoaskSandra,SukeshiorTorrye.DONOTUSETHEIRRADIATORWITHOUTHAVINGSOMEONESHOWYOUHOW.IrradiatecellswiththeIrradiator:Gammacell1,000@TheImmunologyDept.,RoomM-920,DeBakeybuilding,9thfloor(Attention:ChrisArhelger,ifyouhaveanyproblemswiththeIrradiator,contactMs.Arhelger.)oBeforeyouirradiatecells,alwayscheckthattheTurn-tableisON;thatthedosefactorandthetime(inminutes)arecorrect.oDoseFactor=100.0*(October,1998)Time:6MinutesEachminute=1,000rads@thedosefactorof100.Thiswillbeequaltoatotalof6,000rads.11.Aftercellsareirradiated,RETURNEDallthe7x15mlcentrifugetubesto1x50mltube.Dothesameforthesecondsetoftubes.Nowdeterminethetotalcellnumber.12.Determinethetotalcellnumber.Calculatethevolumeofmediarequiredtogiveafinalfreezingdensityof4.2x107cells/ml(reg.Feeders).Eachvial=1ml=4.2x107=10x10cmfeeders.13.Collectthecellsbycentrifugation@1,000rpmfor7minutes.14.Aspirateoffthesupernatantandresuspendthepelletin1/2thevolumecalculated.Usemediaappropriateforthecellsbeingfrozen(i.e.,M15forEScellsor7%FCS,1%GPSforSTO"s).ADDtheSTO誷mediaFIRST.15.Dilutethecellsuspension1:1with2XFreezingMedia(60%DMEM,20%FCS,20%DMSO;freshlyprepared).Addthemediadropwise,mixingwellaftereachaddition.16.Asepticallyaliquotthesuspensionintosterilefreezingvials,labeleachvialwiththefollowing:IRRAD,Dateandcelltype/clonenumber,Passagenumberandplacethevialsintoacryo-freezingcontainerorstyrofoamcontainer.17.Freezethecellsovernight@-70oC,thentransfertothe-135oCfreezerorLiquidNitrogenFreezer.*TheDoseFactorisadequateforonlyoneyear,soannuallytheIrradiatorneedstobecalibrated.(For1996,aftercalibration,thedosefactor=78.0.For1995,dosefactor=71.4.For1994,thedosefactorwas=66.0.FOROCTOBER1998=100.