DifferentiateEScellsintoglialcellsandneurons Day-1:PassEScellsatnormaldensityongelatinizedplatetofreethecultureofcontaminationfibroblastcells. ___________________ Day1:Trypsinizedthecellsasfornormalpassaginguntilthecoloniesliftoff.Trytokeepthelooselyconnectedclumpsofcellstogetherbygentlehandling.Thendirectlyplatethecells1:3intobacterialgradePetridishesinLIFfreemediumcontaining1microMall-transretinoicacid(RA). Day3:Collectcellaggregatesandreplateintissueculturedishes(appr.25cellaggregatesper6cmtissueculturedish)inEScellculturemediumwithoutLIFandRA.Aspiratethemediumcarefully. Day8:Changehalfofthemedium.Fromthisdayonatleast10%ofthecellsexhibitsneuronalphenotype.TheyarespecificallystainedwithCresylVioletandstonglypositivefortheN-CAMantigen.