Notwocelllinesbehaveexactlythesame,soyoumustlearnthepeculiarities,orpersonality,ofeachofthecelllineswithwhichyouwork.Irrespectiveofcelltype,allcellsaredilutedordispersedintoamoresparseculture,andthenallowedtogrowbacktoadenseculture,periodically.Formostcelllines,thisisdonebothMondayandFridayofeachweek.ForsUSPensioncells,allthatyouneedtodoindilutethecellsappropriately.Foradherentcells,youhavetodislodgethemfromthedish,mostoftenbytrypsinization.Howoftendocellsneedtobetransferredandatwhatdensityshouldtheybeseededwhentheyaretransferredorpassaged?Thereisnosimpleanswertothisquestion.Eachcelllineisdifferent.Somecellsdon"tsurviveiftheyareseededattoolowadensity.Somecellsdopoorlywhenallowedtobecometoocrowded.Sometransformedcellswilldepletetheirmediumsoquicklywhentheyaredensethattheybecomesickovernight.Somecelllinesthatexhibitdensity-dependentgrowthinhibitionlosethisiftheyareallowedtoovergrowandbecomedense.Theproblemhereisthatallowing"normal"cellstogrowtoahighdensityselectsforvariantsthatliketogrowwhencrowded.Becausethisisapropertyoftransformedcells,normalcelllinesthatareallowedtogrowathighdensityevolveintopseudo-transformedcelllinesandarenolongersuitableforgrowthregulationstudies.Itisessential,whenworkingwithcellsexhibitinggrowthregulation,totransferthempriortotheirbecomingdense.Lazinessandneglectwillruinsuchcelllines.Somehematopoieticcelllinesthataredependentoncytokinesmustbehandledverycarefully.CTLL-2sareanexampleofthis.IftheyareputintomediumwithaninadequateconcentrationofIL-2,oriftheyuseupalloftheirIL-2,theyimmediatelyundergoapoptosisandcannotberecovered.Thiscanhappeninthespaceof24hours.ThepropertiesofmanyofthecelllinesthatweusearedescribedintheHyperCardCelllinestackthatJamessetup.Adherentcellsmustberemovedfromthedishwhentheyaretobereseeded.Thisusuallyrequirestrypsinization.

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