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1、METHODSMETHODSININMOLECULARMOLECULARBIOLOGYBIOLOGYTMTMVolume290BasicBasicCellCultureCellCultureProtocolsProtocolsTTHIRDHIRDEEDITIONDITIONEditedbyEditedbyCherylD.HelgasonCherylD.HelgasonCindyL.MillerCindyL.MillerBasicCellCultureTechniques11CultureofPrimaryAdherentCellsandaContinuously
2、GrowingNonadherentCellLineCherylD.HelgasonSummaryCellcultureisaninvaluabletoolforinvestigatorsinnumerousfields.Itfacilitatesanalysisofbiologicalpropertiesandprocessesthatarenotreadilyaccessibleattheleveloftheintactorganism.Successfulmaintenanceofcellsinculture,whetherprimaryorimmorta
3、lized,requiresknowledgeandpracticeofafewessentialtechniques.Thepur-poseofthischapteristoexplainthebasicprinciplesofcellcultureusingthemainte-nanceofanonadherentcellline,theP815mousemastocytomacellline,andtheisolationandcultureofadherentprimarymouseembryonicfibroblasts(MEFs)asexamples
4、.Proceduresforthawing,culture,determinationofcellnumbersandviability,andcryopreservationaredescribed.KeyWords:Cellculture;nonadherentcellline;adherentcells;P815;primarymouseembryonicfibroblasts;MEF;hemocytometer;viability;subculturing;cryopreservation.1.IntroductionTherearefourbasicr
5、equirementsforsuccessfulcellculture.Eachofthesewillbebrieflyreviewedinthisintroduction.However,amoredetaileddescriptionisbeyondthescopeofthischapter.Instead,thereaderisreferredtooneofanumberofvaluableresourcesthatprovidetheinformationnecessarytoestablishatissueculturelaboratory,aswel
6、lasdescribethebasicprinciplesofsteriletechnique(1–4).Thefirstnecessityisawell-establishedandproperlyequippedcellculturefacility.Thelevelofbiocontainmentrequired(Levels1–4)isdependentonthetypeofcellsculturedandtheriskthatthesecellsmightcontain,andtransmit,infectiousagents.Forexample,c
7、ultureofprimatecells,transformedhumancelllines,mycoplasma-contaminatedcelllines,andnontestedhumancellsrequireaminimumofaLevel2containmentfacility.AllfacilitiesshouldbeFrom:MethodsinMolecularBiology,vol.290:BasicCellCultureProtocols,ThirdEditionEditedby:C.D.HelgasonandC.L.Miller©Human
8、aPressInc.,T
