basedonsplittingontooneplate

WarmCollagenaseIVsplitmediato37°Cinawaterbath.Aspiratemediaoffofcellcultureplate.Addthefollowingamountofcollagenase:0.5ml/wellof4wellplate1.0ml/wellof6wellplateIncubateat37°Cwith5%CO2for5-10minutes.ThecellsarereadywhentheedgesofthecolonyareroundedupandcurledawayfromtheMEFsontheplate.Usinga5mlpipet,scrapeandwashthecoloniesoffoftheplate.TransfercellsUSPensiontoa15mlconicaltube.Breakupthecoloniesbypipettingupanddownagainstthebottomofthetubeuntilthereappearstobeafinesuspensionofcells(noclumpsofcellsremain).

Spincellsat1000rpmfor5minutes.

Aspiratecollagenaseoffandwashcellswith3mlhumanESmedia.

Spin1000rpm5minutes.

WhilethecellsarespinningpreparethefeederlayersbyaspiratingofftheMEFmediaandwashingonetimewithCa/MgfreePBSandaddinghumanESmediatothefeederlayers(2ml/wellof6wellplate).

Oncecellsaredonespinning,aspirateoffwashmedia.

Resuspendcellsinanappropriatevolume(seenotes).

Platecellsbyadding0.4mlperwellofa6wellplateuntilthelast0.5-0.6mlremain.Addthelastremainingvolumedropwisetoeachwell.

Makesurethecellsareevenlydistributedacrosstheentirewell(seenotes).

Placegentlyinincubator.Againmakesurethecellarenotdisturbed.

Letcellssettleovernightinincubator.

Notes:

AlwayscheckyourMEFsforviABIlityandcontaminationbeforeyousplitontothem.

MEFsareusuallygoodtosplitontoforabout10daysafterplating.

MakesurethatthehumanEScellsareevenlydistributedthroughouttheplate,ifcellsallwindupinthemiddletheycoulddifferentiateorneedtobesplitsooner.

Beverygentletoevenlydistributethecells.Whenusingquickmotions,youwillmostlikelywindupwithyourcellsinthemiddleoftheplate.Whenyouaremovingyourcellsbackandforthintheincubatortodispersethem,besuretoonlymoveinonedirectionatatimeora\"whirlpoollike”effectwillhappenandallofyourcellswillswirlinthemiddle.

ItisagoodideatolearnhumanEScellcultureonMEFsbeforemovingtomatrigel.