ES细胞培养实验方法

ES细胞培育-实验方法EScellculturemediaandsolutionsES细胞培育需要较高的实验条件，培育血清要用纯度较高的（ES级别）的胎牛血清，为防止细胞分化，需要在培育皿底部铺种滋润层细胞（Feedercells）并在培育基中加入白细胞克制因子（LIF）。培育细胞的平皿和吸管均为一次性聚乙烯资料。(A)DMEMwithhighglucose(B)mMnon-essentialaminoacids(100×stock,aliquoted,storedat4℃)(C)1mMsodiumpyruvate(100×stock,aliquoted,storedat4℃)(D)10-4Mβ-mercaptoethanol(100×stock,aliquoted,storedat-20℃)(E)2mML-glutamine(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白细胞克制因子，克制ES细胞分化PreparationofEMFIfeederlayersRegentsFrozenvialsofprimaryembryofibroblastsTissueculturedishesPBSwithoutCa2+andMg2+tripsininsaline/EDTADMEM+10%FBSMitomycinC(stock1mg/mlinPBSstoredindarkat4℃andusedwithintwoweeks,mitomycinCistoxic;wearglovesandusecautionwhenhandling)Methods1.ThawafrozenvialEMFIcellsquicklyat37℃.g,5min)2.Addcellsto10mlDMEM+10%FBSandcentrifuge(270Decantsupernatant,resuspendthecellpelletgentlyin10mlDMEM+10%FBS,andsplitontofive150mmplateseachcontainingatotalof25mlDMEM+10%FBS.Mixwell.注意摇动混匀，不要纯真只按一个方向摇动，以使细胞较平均地散布于平皿中，。4.Incubatecellsat37℃,5%CO2.Whenthecellsformaconfluentmonolayer(approx.threedays)eachplateshouldeitherbe:(a)Thrypsinized,splitontofiveadditional150mmdishes,andgrownuntiltheyformaconfluentmonolayer,or(b)DirectlytreatedwithmitomycinC（丝裂霉素）toinhibitcellgrowthanddivision.由于ES细胞与滋润细胞共培育时，未经丝裂霉素办理的滋润细胞增殖很快，会与ES细胞竞争养分，此步丝裂霉素办理可使滋润细胞失掉增殖，但仍保持存活。Removethemediumfromtheconfluentplatesand10mlDMEM+10%FBScontaining100μlmitomycinC(1mg/mlstock).Swirlplatestoensureanevendistributionofmedium.7.Incubatecellsat372℃,5%COfor.8.Washthemonolayerofcellstwicewith10mlPBSperdish.9.Add5mltrypsin/EDTAtoeachplate.10.2incubate37℃,5%COuntilthecellscomeofftheplate.11.Add10mlDMEM+10%FBStoeachplateandbreakanycellaggregatesbygentlypipetting.Centrifugecells(270g,5min)andresuspendthepelletinDMEM+10%FBS.13.Countthecellsanddilutetoaconcentrationof2×105cells/ml.PlatethecellsimmediatelyontotissueculturedishescontainingDMEM+10%FBSfortheappropriatecelldensitiesandvolumesofmediumfordifferentplatesizes.15.Allowfeederstoattachatleast2h,butpreferablyovernight,beforeaddingEScells.ChangethemediumtoEScellmediumimmediatelybeforeaddingEScells.MitomycinCtreatedEMFIfeederscanbeusedforuptosevendayswithmediumchangeseverythreetofourdays.PreparatiooonofastockofmitomycinCtreatedEMFIcellsReagents正常的滋润细胞贴壁生擅长培育皿底面，呈梭形，应该平均散布，并将皿底完整覆盖。1.thawafrozenviaofEMFIcellsquicklyat37℃.Addcellsto10mlDMEM+10%FBSandcentrifuge(270g,5min).Decantsupernatant,resuspendthecellpelletgentlyin10mlDMEM+10%FBS,andsplitontofive150mmplateseachcontainingatotalof25mlDMEM+10%FBS.Mixwell.4.incubatecellsat37℃,5%CO2.5.Whenthecellsformaconfluentmonolayer(approx.threedays)eachplateshouldtrypsinized,splitontofiveadditional150mmdishes,andgrownuntiltheyformaconfluentmonolayer6.Removethemediumfromtheconfluentplatesand10mlDMEM+10%FBScontaining100μlmitomycinC(1mg/mlstock).Swirlplatestoensureanevendistributionofmedium.7.Incubatecellsat37℃,5%CO2for.Washthemonolayerofcellstwicewith10mlPBSperdish.Add5mltrypsin/EDTAtoeachplate.10.incubate37℃,5%CO2untilthecellscomeofftheplate.(5-10min).11.Add10mlDMEM+10%FBStoeachplateandbreakanycellaggregatesbygentlypipetting.12.Centrifugecells(270g,5min)andresuspendthepelletinfreezingmedium.Freezingallthecellsfromeachplateinonefreezingvialin1mlof1×freezingmediumandstoreat-70℃foroneday.Transferthevialstoliquidnitrogen.13.tomakefeederplatesthawafrozenvialofmitomycinCtreatedEMFIcellsquicklyat37℃14.Addcellsto10mlDMEM+10%FBSandcenrifuge(270g,5min).15.Decantsupernatant,andresuspendthecellpelletgentlyin30mlDMEM+10%FBS.oftheplatesrequiredput10ml/100mmplate,5ml/60mmplate,5ml/60mmplate,or35mmplate.17.Allowfeederstoattachpreferablyovernight,beforeaddingEScellsoratleast2hifusinggelatinizedplates.18.ChangethemediumtoEScellmediumimmediatelybeforeaddingEScells.GrowthofEScellsonfeedersplatesEquipmentandreagents100mmdishescontainingfeederlayersEScellmediumbrieflypre-warmedto37℃0.05%trypsininsaline/EDTAPBSwithoutCa2+andMg2+Gelatin,%solutioninwater,autoclavedMethod1.QuicklythawonevialoffrozenESbywarminginyourhandorat37℃,andtransfercellstoa12mltubecontaining10mlofEScellmediumbeforealltheicehasdisappeared.2.Centrifugeat270gfor5min.3.Aspiratesupernatantandresuspendpelletin10mlEScellmediumandplateona100mmdishwithafeederlayer.4.Changethemediumthenextdaybyswirlingthemediumindishtocollectdebris,thenaspirate.Addfreshmediumgentlytothesideoftheplatesothatthefeederlayerisnotdisturbed.5.Onthesecondday(cellshouldbejustsubconfluent)washcellstwicewithPBSandadd2mltrypsin/EDTA.6.Incubateforapprox.5minat37℃untilcellsbegintocomeofftheplate.Gentlyagitatetheplateandobserveunderamicroscope.Whentrypsinizationiscomplete,cellsshoulddetachassmallclumps,notasasinglesheetorsinglecells.Add5mlEScellmediumandgentlypipettethecellsupanddowntobreakcellsclumps,Ifthecellsaresticky,gentlypipettebeforeaddingmedium.Transfertoasterile12mltubeandpelletcellsincentrifuge(5min,270g).10.Aspiratesupernatantandgentlyresuspendcellpelletin5-7mlmedium.×10611.Add1mlofthecellsuspention(about2-5cells)toafresh100mmfeederlayerdishcontaining9mlEScellmedium.DisperseEScellsevenlybypipettinggentlyandrockingtheplatepriortoincubatingat37℃，5%CO2。12.Changethemediumthenextdayandpasscellseveryseconddayasdescribedabove.正常的未分化的ES细胞应该呈球状细胞团附着于滋润细胞层表面，细胞团的边缘与滋润细胞层因有空间距离而形成折光的亮边，假如细胞有分化，则细胞团的球形边沿部分或所有平展化，与基层滋润细胞之间的亮边消逝，分化了的细胞不该再使用。LongtermfreezingofEScellstocksMethod短期保留能够放在-70℃，但长久保留应转入液氮。1.Trypsinizecellsfroma100mmdish.2.Pelletcellsbycentrifugationandrespensionin3mlprecooledfreezingmedium.Immediatelyaliquot1mlofthecellsuspentionintofreezingvialsonice.4.ImmediatelytransfervialtoaStyrofoamboxpre-cooledto-70℃,orslow-coolcontainer,andthenintoa-70℃frezer.Itisimportanttoworkquickly.5.After24htransferthetubesondryicetoliquidnitrogen.SandardelectroporationEScellsEuipmentandreagentsElectroporationapparatus:Bio-RadGenePulserandCapacitanceExtenderEScellmedium2+2+PBSwithoutCaandMgGeneticin(G418)OntheseconddayafterpassingrecentlythawedEScells,trypsinizecells,buttrypsinizeforlongertoobtainsinglecells.Afterpipettingthecellsgentlyin3mltrypsintobreakuptheclumps,add7mlmediumandpipettegentlyupanddownagain.Pre-platethecellsbyincubatingtheminthedishesfor15-20mintoallowfeedercellstore–attachtotheplate.HarvesttheEScellsbycarefullymixingandwithdrawingmedium,andtransfercellstoa50mlFalcontube,combiningthecellsfromtwotofiveplate.Pelletthecellsfor5minat270gAspiratethesupernatantandresuspendthecellsinaminimalvolumeofPBS(～1ml/100mmplatestartingculture).Keepcellsonice.7.CounttheESCellsusingahaemocytometerandadjustcellconcentrationto7-10×106cells/mlwithsterilePBS.8.Mixmlofthecellsuspentionwith25-40μgoflinearizedvectorDNA,andtransferintoanelectroprationcavettewhichhasbeenpre-cooledonice.9.Setuptheelectroporationconditionsinadvance:240V,500μFfortheBio-RadGenePulserusingtheCapacitanceExtender.10.Transfereachcuvetteholderwithelectrodesfacingtheoutputtheelectricpulse.11.Removeeachcuvettefromthecuvetteholderandplaceonicefor20min.12.RemovethecellsfromeachcuvetteanddiluteinappropriatevolumeofEScellmedium(15-20ml/cuvette).Cellsfromseveralcuvettescanbecombined.13.Transfer10mlresuspendedEScells.Dispersecellsevenlybyrockingtheplate.14.Changemediumthenextmorning.15.Twodaysafertheelectroporation,begindrugselection16.Changethemediumeverydayifworkingwithgancyclvir,sinceitcanbreakdownandbecometoxictothecells,otherwiseeverytwodayswhenusingG418selectiononly.Widespreadcelldeathshouldbeapparentafertwotothreedaysofdrugselection.首次采纳的G418的浓度为200ug/ml,固然也能看到大量的细胞死亡，但阴性比较（不加DNA而用TE的电转）亦见大量细胞死亡。17.Afteraboutsixtoeightdaysofselection,individualdrug–resistantcoloniesshouldhaveappearedandbelargeenoughtopickandsubcloneforscreeningbySouthernblotanalysisorbyPCRandtofreezeforstorage.GrowingdrugresistantclonesMethod1.Preparetwosetsof96-wellplates,onecontaining35μloftrypsin/EDTAandonegelatincoated.我们没有明胶办理的培育皿，而是早先在皿中铺种滋润细胞层，但滋润细胞过于稀少，造成ES细胞发生疏化，此后实验应该防止，应使滋润细胞浓密完整2.CircleallvisibleEScellcoloniesthatwillbepickedonthebottomeachplatewithamarkerbyholdingplateuptolightorusinganinvertedmicroscope.WashEScellcontainingplatestwicewithPBS.LeavecellsinPBSduringpicking.Afterpicking,replacePBSwithESmediumandreturnplatetoincubatorifadditional,smallercolonieswillbepickedonsubsequentdays.Pickthemmdrug–resistantEScellclonesthatformafterabouteightdaysofselectionwithadrawn-outPasteurpipetteorayellowGilsontipunder℃adissectingmicroscope.Pickcoloniesofsimilarsize,ortakeanequivalentportionoflargecolonies.挑取细胞克隆时将细胞间的显微镜置于超净台中，一半镜身（物镜）在隔绝玻璃里面，一半镜身（目镜）在外面，紫外照耀消毒。操作时需要几个人，以加迅速度。注意ES细胞的形态应该保持球形细胞团，而发生疏化的细胞克隆应该舍弃。TransfereachcolonyinaminimalvolumeofPBSintoonewellofaVshaped96-wellplatecontaining35μlof%trypsininsaline/EDTAatroomtemperature.Usuallyeveryotherrowisusedand48coloniespickedatonetime.6.Afterpicking48colones,incubateplateforminat37℃,5%CO2,untilcellclumpsbreaksup.7.Stopthereactionbyadding100μlofESmediumcontainingG418toeachwellusingmultichannelpipettor.G418的浓度宜提升至400-600ug/ml.8.Mixcellsgentlybypipettingupanddownandtransfertogelatinized96-welltissuecultureplate.9.WasheachV-wellfromthetrpsinplatewithanother100μlofmediumperwellandaddtosamewellofthe96-wellgelatinizedplate.10.Changethemediumthenextday,andthendailythereafter.11.Ifthecolonieshavenotgrownto～80%confluenceintwotothreedays,tryplatethecellsusingthefollowingprocedure:(a)AspiratethemediumandwashthecellswithPBS.(b)Add35μloftrypsin/EDTAandincubatefor5minat37℃,oruntilthecellsliftoffthedish.(c)Add200μlEScellmediumandbreakcoloniesupbygentlypipettingupanddown.(d)Changethemedium12-24hlater.12.Repeatsteps1-11onsecondandthirdpickingdays,usingoriginalplatesofelectroporatedcells.13.Whenthecoloniesin96-wellplateshavereachatleast80%confluenceyoushouldpassagethemandfreezeyourclonesandpreparereplicaforDNAanalysis.Freezingdrugresistantclonesandpreparationofreplica