ES细胞培养-实验方法..docVIP

v1.0 可编辑可修改 ES细胞培养 - 实验方法 ES cell culture media and solutions ES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（ Feeder cells ）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) DMEM with high glucose (B) mM non-essential amino acids (100 × stock,aliquoted ,stored at 4 ℃) (C) 1 mM sodium pyruvate (100 ×stock,aliquoted ,stored at 4 ℃ ) (D) 10-4 M β- mercaptoethanol (100 × stock,aliquoted ,stored at-20 ℃) (E) 2mM L- glutamine (100 ×stock,aliquoted ,stored at-20 ℃) (F) 15%FBS要用纯度较高的（ ES级别）的胎牛血清 (G) Penicillin and streptomycin (final concentration 50 μ g/ml each) (H) 1000 U/ml LIF 白细胞抑制因子，抑制 ES 细胞分化 Preparation of EMFI feeder layers Regents Frozen vials of primary embryo fibroblasts Tissue culture dishes PBS without Ca2+ and Mg 2+ % tripsin in saline /EDTA DMEM +10%FBS Mitomycin C (stock 1 mg/ml in PBS stored in dark at 4 ℃ and used within two weeks ,mitomycin C is toxic ;wear gloves and use caution when handling ) Methods 1 v1.0 可编辑可修改 1. Thaw a frozen vial EMFI cells quickly at 37 ℃ . 2. Add cells to 10 ml DMEM +10%FBS and centrifuge (270 g, 5 min) Decant supernatant , resuspend the cell pellet gently in 10 ml DMEM +10%FBS, andsplit onto five 150 mm plates each containing a total of 25 ml DMEM+10%FBS.Mix well. 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. Incubate cells at 37 ℃ ,5% CO 2 . When the cells form a confluent monolayer (approx. three days )each plate should either be: (a)Thrypsinized ,split onto five additional 150 mmdishes ,and grown until they form a confluent monolayer ,or (b)Directly treated with mitomycin C （丝裂霉素） to inhibit cell growth and division . 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。 Remove the medium from the confluent plates and 10 ml DMEM + 10%FBS containing 100 μ l mitomycin C (1mg/ml stock).Swirl plates to ensure an even distribution of medium. 7. Incubate cells at 37 2 ℃ ,5% CO for . 8. Wash the monolayer of cells twice with 10 ml PBS per dish . 9. Add 5 ml trypsin /EDTA to