Youwillneed:

13.5daypregnantmouse(weuseMTKNEOinbredwhitemice)

2setssterileinstruments

onecontainingapairofcurvedforcepsandapairofirisscissors

onecontainingtwopairscurvedforceps,onepairirisscissorsanda#3sizescalpelhandle

Phosphatebuffersaline(PBS)

Sterilemediumsizepetridishes(tissueculturestandard)

18gaugeneedle

luerlocksyringe(about6ccshouldsuffice)

#11sizeflat-edgedscapleblade

trypsin/EDTA

Dulbecco"sModificationofEaglesMedium(with10%fetalcalfserum,1%penicillin/streptomycin,1%L-glutamine0.2%0.1mBME)

largeflasks(tissueculturedstandard-about154cm2area)

ClassIILaminarFlowhood

Beforestarting,pourout2xpetridishesofPBSinthehood.Pregnantmouseiskilledbycervicaldislocation.(Thisisnotdoneinthehoodbutoncleanbenchcote).Laymouseoutonitsbackandswabbellywith70%ethanol.Withapairofscissors(notsterile)nipasmallcutacrossthebelly.Graspingtheskinaboveandbelowthenipwithyourfinger,teartheskinapartanddrawbackovertheheadandhindlegstoexposethevisceraofthegut.Thismethodiscleanerthancuttingthroughthefurandenableyoutoreachtheuteruswithnoriskoftouchingthefur(cuttingthroughdryfurcreatesabacterialaerosol).

Usingsterileforcepsandirisscissorsdissectouttheuterus,takingcarenottotouchthefurorthebenchcotewiththeuterusorinstruments.PlacetheuterusintoapetridishofsterilePBSandswirlaroundtoremoveblood.TransferuterustosecondpetridishofsterilePBSandmovedishtohood.

Usingthesecondsetofsterileinstrumentsandafreshsterilepetridish,isolatetheembryos.Besuretoremovetheplacementandembryonicsacs.Usingthescalpelhandlewiththe#11bladeonit,cutoffembryoheadsandscoopouttheliverwithapairofforceps.Theheadandforelimbshouldbecutoffasshownbelow.

DiscardtheheadandliverandleavethebodiesinfreshPBSinafreshpetridish.

Take6ccluerlocksyringewith18gaugeneedleattachedandremoveplunger.KEEPPLUNGERSTERILE.Droptheembryobodiesinsidethesyringeandadd3mltyrpsin/EDTA.Putplungerbackinsyringeandsquirtcontentsofsyringeintoalargetissuecultureflask.Placetheflaskontoawarmingtray(37¡C)for2-3minutes.Backtothehoodandadd20mlDMEM.WhenaddingtheDMEM,trytowashanytissueoffthewallsoftheflask.Pipettethetissue/mediumupanddownafewtimestohelpbreakupthetissue.Transferflasktoanincubatorat37¡Cwith5%CO2.Donotputlessthan7embryoinonelargeflask.

IMPORTANT:Loosenlidofflaskinincubatortoallowgasexchangeinmedium.

Thisistheprimaryisolationorpassageone.PMEF"sshouldattachandbegintodividein1-3days.Duringthistimedonotdisturb,soastoallowPMEF"stosettleandattach.

After2dayschangethemedium.Itwillbeveryacidic.After3-4daystheculturewillneedsplitting.Removemediaandgentlywashthemonolayerwith2X10mlPBS.Add2mltrypsinEDTAandsplit1:4.Afterafurther2-4daystheculutrewillbereadyforfreezing.Thenumberobtainedfromeachflaskwillbebetween5-10x106cells.Freezecellsin10%DMSOat3X106/ampule.

WhenrecoveringthecellsfromLN2putallthecellsintoamediumflask.Whenconfluentthesearesplitinto1mediumandonelargeflask(1:3).ThelargeflaskcanbetreatedwithmitomycinCandthemediumflasksplitagain.DonotpassagebeyondP6.