hct 116、hela、MEFs细胞参数介绍.doc

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1、HELAHeLa(ATCC® CCL-2™)ATCC：Hela细胞，源于黑人31岁女性，子宫颈腺癌，是一种附着型上皮细胞，要求存于液氮中。Thesecellsareasuitabletransfectionhost.Thiscelllinecanbeusedtoscreenfor Escherichiacoli strainswithinvasivepotential.BiosafetyLevel：（2[Cellscontainhumanpapillomavirus]BiosafetyclassificationisbasedonU.S

2、.PublicHealthServiceGuidelines,itistheresponsibilityofthecustomertoensurethattheirfacilitiescomplywithbiosafetyregulationsfortheirowncountry.）CompleteGrowthMedium：ThebasemediumforthiscelllineisATCC-formulatedEagle'sMinimumEssentialMedium,CatalogNo.30-2003.Tomakethecomple

3、tegrowthmedium,addthefollowingcomponentstothebasemedium:fetalbovineserumtoafinalconcentrationof10%.Subculturing（接种）：Volumesusedinthisprotocolarefora75cm2flask;proportionallyreduceorincreaseamountofdissociationmediumforculturevesselsofothersizes.CorningT-75flasks(catalog#

4、430641)arerecommendedforsubculturingthisproduct.1.Removeanddiscardculturemedium.2.Brieflyrinsethecelllayerwith0.25%(w/v)Trypsin-0.53mMEDTAsolutiontoremovealltracesofserumwhichcontainstrypsininhibitor.1.Add2.0to3.0mLofTrypsin-EDTAsolutiontoflaskandobservecellsunderaninver

5、tedmicroscopeuntilcelllayerisdispersed(usuallywithin5to15minutes).Note:Toavoidclumpingdonotagitatethecellsbyhittingorshakingtheflaskwhilewaitingforthecellstodetach.Cellsthataredifficulttodetachmaybeplacedat37Ctofacilitatedispersal.2.Add6.0to8.0mLofcompletegrowthmediumand

6、aspiratecellsbygentlypipetting.3.Addappropriatealiquotsofthecellsuspensiontonewculturevessels.4.Incubateculturesat37C.SubcultivationRatio:Asubcultivationratioof1:2to1:6isrecommendedMediumRenewal:2to3timesperweekCryopreservationFreezeMedium: Completegrowthmediumsupplement

7、edwith5%(v/v)DMSOStorageTemperature: LiquidnitrogenvaporphaseCultureConditionsAtmosphere: Air,95%;carbondioxide(CO2),5%Temperature: 37°C论坛：贴壁生长，铺路石状，长得快，2-3天传代一次，传代不及时会造成老化的细胞堆积，看起来很脏。10%+1640或者10%+高糖DMEM都有培养。MEFs_283TAg(ATCC® CRL-2822™)OrganismMusmusculus,mouseTissueemb

8、ryoCellTypefibroblastimmortalizedwithSV40largeTantigenSV40largeTantigentransfectedProductFormatfrozenMo