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1、ES细胞培养-实验方法EScellculturemediaandsolutionsES细胞培养需要较高的实验条件，培养血清要用纯度较高的（ES级别）的胎牛血清，为防止细胞分化，需要在培养皿底部铺种滋养层细胞（Feedercells）并在培养基中加入白细胞抑制因子（LIF）。培养细胞的平皿和吸管均为一次性聚乙烯材料。(A)DMEMwithhighglucose(B)0.1mMnon-essentialaminoacids(100×stock,aliquoted,storedat4℃)(C)1mMsodiumpyruvate(100×stock,
2、aliquoted,storedat4℃)(D)10-4Mβmercaptoethanol(100×stock,aliquoted,storedat-20℃)(E)2mMLglutamine(100×stock,aliquoted,storedat-20℃)(F)15%FBS要用纯度较高的（ES级别）的胎牛血清(G)Penicillinandstreptomycin(finalconcentration50μg/mleach)(H)1000U/mlLIF白细胞抑制因子，抑制ES细胞分化PreparationofEMFIfeederlayers
3、RegentsFrozenvialsofprimaryembryofibroblastsTissueculturedishesPBSwithoutCa2+andMg2+0.05%tripsininsaline/EDTADMEM+10%FBSMitomycinC(stock1mg/mlinPBSstoredindarkat4℃andusedwithintwoweeks,mitomycinCistoxic;wearglovesandusecautionwhenhandling)Methods1.ThawafrozenvialEMFIcellsqu
4、icklyat37℃.2.Addcellsto10mlDMEM+10%FBSandcentrifuge(270g,5min)3.Decantsupernatant,resuspendthecellpelletgentlyin10mlDMEM+10%FBS,andsplitontofive150mmplateseachcontainingatotalof25mlDMEM+10%FBS.Mixwell.注意摇动混匀，不要单纯只按一个方向摇动，以使细胞较均匀地分布于平皿中，。4.Incubatecellsat37℃,5%CO2.5.Whenthec
5、ellsformaconfluentmonolayer(approx.threedays)eachplateshouldeitherbe:(a)Thrypsinized,splitontofiveadditional150mmdishes,andgrownuntiltheyformaconfluentmonolayer,or(b)DirectlytreatedwithmitomycinC（丝裂霉素）toinhibitcellgrowthanddivision.因为ES细胞与滋养细胞共培养时，未经丝裂霉素处理的滋养细胞增殖很快，会与ES细胞竞争
6、养分，此步丝裂霉素处理可使滋养细胞失去增殖，但仍保持存活。1.Removethemediumfromtheconfluentplatesand10mlDMEM+10%FBScontaining100μlmitomycinC(1mg/mlstock).Swirlplatestoensureanevendistributionofmedium.2.Incubatecellsat37℃,5%CO2for2-2.5h.3.Washthemonolayerofcellstwicewith10mlPBSperdish.4.Add5mltrypsin/ED
7、TAtoeachplate.5.incubate37℃,5%CO2untilthecellscomeofftheplate.6.Add10mlDMEM+10%FBStoeachplateandbreakanycellaggregatesbygentlypipetting.7.Centrifugecells(270g,5min)andresuspendthepelletinDMEM+10%FBS.8.Countthecellsanddilutetoaconcentrationof2×105cells/ml.9.Platethecellsimme
8、diatelyontotissueculturedishescontainingDMEM+10%FBSfortheappropriatecelldensitiesa
