mcf-7细胞的培养传代

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1、1.MCF-7细胞的培养传代细胞株购自武汉中国典藏物培养中心（美国ATCC株亚型），培养基是MEM，10％的小牛血清（购自杭州四季青公司）。传代要看细胞数的多少，一般来说10％的细胞，要5天左右，如果90％融合的细胞一传2，3天子代细胞即可传代。传代：偶用的是25ml培养瓶，吸出培养基，6mlpbs冲洗3次，加入0.25％的胰酶2－3ml，胰酶均匀盖满瓶壁，消化2－5min左右（可以同时振摇），细胞缩起变圆，浮起，加入培养基2－3ml，不用太精确，吹打成单细胞悬液分瓶即可。室温下消化即可，因为是成纤维细胞，较为耐受胰酶，消化时间稍长亦可，要注意mcf-7细胞是一种肿瘤细胞，生长规律

2、性差，很容易不均匀，要注意吹打的充分性。摘自ATCCOrgan:mammarygland;breastCellularproducts:insulin-likegrowthfactorbindingproteins(IGFBP)BP-2;BP-4;BP-5Completegrowthmedium:ThebasemediumforthiscelllineisATCC-formulatedEagle'sMinimumEssentialMedium,CatalogNo.30-2003.Tomakethecompletegrowthmedium,addthefollowingcompone

3、ntstothebasemedium:0.01mg/mlbovineinsulin;fetalbovineserumtoafinalconcentrationof10%.Atmosphere:air,95%;carbondioxide(CO2),5%Temperature:37.0°CSubculturing：1.Removeculturemediumtoacentrifugetube.2.Brieflyrinsethecelllayerwith0.25%(w/v)Trypsin-0.53mMEDTA(0.015%,w/v)solutiontoremovealltracesofse

4、rumwhichcontainstrypsininhibitor.3.Add2.0to3.0mlofTrypsin-EDTAsolutiontoflaskandobservecellsunderaninvertedmicroscopeuntilcelllayerisdispersed(usuallywithin5to15minutes).Note:Toavoidclumpingdonotagitatethecellsbyhittingorshakingtheflaskwhilewaitingforthecellstodetach.Cellsthataredifficulttodet

5、achmaybeplacedat37Ctofacilitatedispersal.4.Add6.0to8.0mlofcompletegrowthmediumandaspiratecellsbygentlypipetting.5.Transferthecellsuspensiontothecentrifugetubewiththemediumandcellsfromstep1,andcentrifugeatapproximately125xgfor5to10minutes.Discardthesupernatant.6.Resuspendthecellpelletinfreshgro

6、wthmedium.Addappropriatealiquotsofthecellsuspensiontonewculturevessels.7.Incubateculturesat37C.SubcultivationRatio:Asubcultivationratioof1:3to1:6isrecommendedMediumRenewal:2to3timesperweekFreezemedium:Completegrowthmediumsupplementedwith5%(v/v)DMSOStoragetemperature:liquidnitrogenvaporphase注（英

7、文部分摘自ATCC）：1.0.25%Trypsin-0.53mMEDTA：0.25%Trypsin/0.53mMEDTAinHanksBalancedSaltSolutionwithoutcalciumormagnesium.Fordissociationofcellmonolayers.Trypsin-EDTAsolutionissuitableformostbutnotforalladherentcelllines.2.FetalBovineSerumStorag