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1、TechniquesforMammalianCellAPPENDIX3FTissueCultureTissueculturetechnologyhasfoundwideapplicationinthefieldofmolecularbiology(see,inparticular,Chapter9,IntroductionofDNAintoMammalianCells,andChapter16,ProteinExpression).Monolayercellculturesareutilizedinc
2、ytogenetic,biochemical,andmolecularlaboratoriesforresearchaswellasdiagnosticstudies.Inmostcases,cellsortissuesmustbegrowninculturefordaysorweekstoobtainsufficientnumbersofcellsforanalysis.Maintenanceofcellsinlong-termculturerequiresstrictadherencetoasep
3、tictechniquetoavoidcontaminationandpotentiallossofvaluablecelllines.Thefirstsectionofthisunitdiscussesbasicprinciplesofsteriletechnique.Animportantfactorinfluencingthegrowthofcellsincultureisthechoiceoftissueculturemedium.Manydifferentrecipesfortissuecu
4、lturemediaareavailableandeachlaboratorymustdeterminewhichmediumbestsuitstheirneeds.Individuallaboratoriesmayelecttousecommerciallypreparedmediumorpreparetheirown.Commerciallyavailablemediumcanbeobtainedasasterileandready-to-useliquid,inaconcentratedliqu
5、idform,orinapowderedform.Besidesprovidingnutrientsforgrowingcells,mediumisgenerallysupplementedwithantibiotics,fungicides,orbothtoinhibitcontamination.Thesecondsectionofthisunitdiscussesmediumpreparation(seealsotheintroductiontoChapter9).Ascellsgrowninm
6、onolayerreachconfluency,theymustbesubculturedorpassaged.Failuretosubcultureconfluentcellsresultsinreducedmitoticindexandeventuallyincelldeath.Thefirststepinsubculturingistodetachcellsfromthesurfaceoftheprimaryculturevesselbytrypsinizationormechanicalmea
7、ns.Theresultantcellsuspensionisthensubdivided,orreseeded,intofreshcultures.Secondaryculturesarecheckedforgrowth,fedperiodically,andmaybesubsequentlysubculturedtoproducetertiarycultures,etc.Thetimebetweenpassagingcellsvarieswiththecelllineanddependsonthe
8、growthrate.TheBasicProtocoldescribessubculturingofamonolayerculturegrowninpetriplatesorflasks.SupportProtocolsdescribefreezingofmonolayercells,thawingandrecoveryofcells,countingcellsusingahemacytometer,andpreparingcellsfortranspo
