S细胞培养实验措施ES cl cuturemedia and soluinS细胞培养需要较高旳实验条件，培养血清要用纯度较高旳（E级别）旳胎牛血清，为避免细胞分化，需要在培养皿底部铺种滋养层细胞(Feedr cls）并在培养基中加入白细胞克制因子（IF)。培养细胞旳平皿和吸管均为一次性聚乙烯材料。(A) DMEM t gh ucos (B) 0.mM nonestial aminods （00tock，aliqted ,storedat 4)(C) 1 mM odum pyruvat (0so,luoted ,srat 4)(D) 1-4M meratoetanol （100stock,aute ,store a-2）(E) 2mM glmne (10tock,liquotd,sored at2)(F) 15%FBS要用纯度较高旳（E级别)旳胎牛血清(G) Penillin and sreptyci(finl concetrton50 g/mlah)(H) 100 Uml LIF 白细胞克制因子，克制ES 细胞分化 Prprt o EMI feder lyrs egens Frozen alsf rim ebyofirobasts Tissue cultre dishs PBS wihota2+an M2+0.05% tripsin in alin /EDTADMEM+10%FBSMitomyci C(ock mg/ml in BSsoredin arkat and sedwhi wo weeks ,miycn Ci oxic ;wear gloes nuse autin when haig ）Mehd1. ha frozen viaEFels qickly at 3.2. Addclls 10 m MEM +1%FB and cerifu (20g,5 min）3. eantsupenatant , resuspndhe cell peet entl in1 m DMM +10%FBS， ndplit nto fie15 mmpatseah cntanig atota of25m DMM +10FBSM well.注意摇动混匀,不要单纯只按一种方向摇动,以使细胞较均匀地分布于平皿中，。4. ubt cellsat 37 ,5% C2 5. Whe th cells orm a conlentmonolaye(aprx. three das ）ehplateshould iter be：()Thrypinzed ，sp ont ive addtiona 50 mmdishe ，nd gwn until ty fra conflun mnolae , (b)Direcytrated with miomycin C（丝裂霉素）t nit celrwthnddiion .由于ES细胞与滋养细胞共培养时,未经丝裂霉素解决旳滋养细胞增殖不久,会与S细胞竞争养分，此步丝裂霉素解决可使滋养细胞失去增殖,但仍保持存活。6. Removethe mdimfrom the conle ps and 10 mlDMEM+1FBS contining 10 mtoycin C (1mgmlstoc).Swil lates o ensure aneveistriutifim.7. Inae cells a37,%O2 fo 2-25h.8. Wash te mnoayer fcelltwic wth 0 ml PBSperdish .9. dd ml trpin EDTA t eachplae 10. incbat 7，5%CO2 ntil the l come off the plate 11. Ad 10 ml DMEM+1FBS to ech plae adreaan el agregatesby genty pipettin.12. Cenrifuge ells (20 g,5min)and resusenthe llet n DMEM+1%FBS13. Cout th cells ndilute tacentraion of 2 10cl/ml.14. lae th ce imeditely onotissueculture dishes contninME+0%FB or he approriae ell densties a volume of medim r ifferen plate ses.15. Allow fers t attch at eat 2 ，but prefab overnigt ,befre adding ES cls.16. anethe medium oESellmdimediatel oeaing S cells .MiomycinC taedEMFfede canbeused f upto seven dys with mediu changes every thre to four aysPreratoonof a stokofmtomycnC treated MI cls Reaents正常旳滋养细胞贴壁生长于培养皿底面，呈梭形,应当均匀分布，并将皿底完全覆盖。1. haw afozen va of FI cls quckly a7.2. Add cls to 10 ml DME 10%FBS a ntrifue (20 g,5min)3. Dcantsuerntat ， resuspendhe cell pellegenl in10 ml EM +0%FBS， and spli onofie 50 mm paes each contaiig a ttal of 2 ml DEM 10%F Mix wel.4. incubtecells at 37,5%CO . 5. Whe th cells frm conluent mooyer (apprx. hredas)ch pte shld rypszed ,splitnto fve addiional 15 dihes,d rown unl they orm confuent mnolayer 6. Rmoe he medu fro th cnfunt ptean 10 ml DMM + 10%BS onain 00l mitomyin C(1mgml soc）.Swirlpt tenua even istriuto of medi7. Inbate cls at 37，5% C2 for 2-.5h8. Wash the mnlaer f cells twic wth 10 ml PBS p ds 9. Ad5l trypsin /EDA to each plate .10. incubate 7,5% O nil te els com of e ple .(10min）11. Ad 10 ml DMEM 10BSto ec pltend break any cell gratsy genly ipetting.12. etifue cells (7,5min）andrsuspend the elle freengediu .Freezing al the cell from ech a ioe feezin vial in 1l of freezng medim and ore t - fo day Transfr the vias t u tro.13. to make eer plats ta a frozevial ofmtomcn C treae MI cellsquikly a 314. Addcelsto 1 ml DM +1%BSnd ciuge （270 ,5min)15. Dect superntat,and rspend h cel pelltgnty n0mlMM +0%FBS.16. ecells dectly ito tissuecultre pat.Depenng ofthe ze ofth pltesrequired pu 1 ml /100mm, 5l /60 plt,5/60 mm pte, r1.5ml/mm la.17. Alow feedrs to atachpreeabl vernight ，befoe ding ES elso at least 2h i usig lainizedpates 18. Chnete dium toS ce medium miately beforaddin ESell. Growthf ESclsn feedersplatesEqipnt ad reagens 100mm dishes ontaiingfeder ayers ES ell mediu briefly pr-wrmedo 30. 0% trysinin sain EDTAPBS wittCa nd Mg2Gean ,0.1% soluio in water ，autoclavedMd