《NBA2K16》全面补ID及对应球员一览_面补ID（1）游民 ...
来自 : 蚂蚁淘
LethumanEScellsgrowuntilthecoloniesarelargeandthecellsareprettypiledup-aboutthetimewhenyouwouldnormallysplitorevenadaypastthat.Treatcellswith0.2-0.5mg/mldispase.YouwanttousethelowestpossIBLeconcentrationofdispase,butittendstovaryabit.Waituntilthecoloniescompletelydetachfromtheplate.Donotblowcoloniesoffwithapipet.Thisshouldtakeabout20-30min.Ifnothingishappeningbythatpoint,addmoredispase.Oncethecoloniescomeup,gentlytransferthemtoa15mlconicaltubewitha10mlpipet.Youdon"twanttobreakupthecolonies.Thecellsshouldsinktothebottomofthetubeafteraminuteortwowithoutanyspinning.AspirateoffthemediaandwashonceinhESmedia.Ifyouareinahurryandneedtospinthecoloniesdown,1minat500rpmisenough.TransfercellstoaflaskcontainingESmediawithoutbFGF.PutalloftheEBsfromone6-wellplateintoaT80flaskwithabout25mlsmedia.Thecellswillroundupintoactualembryoidbodiesafterabout12-24hrs.Theyshouldthenbefedeverydaybyexchanginghalfthemediawithfreshmedia.TheEBsshouldnotattach,iftheydo,gentlytaptheflasktodislodgetheEBs.
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