basedonsplittingontoonplate WarmCollagenasemediato37°Cinawaterbath. Aspiratemediaoffofcellcultureplate. Addthefollowingamountofcollagenase 0.5ml/wellof4wellplate 1.0ml/wellof6wellplate Incubateat37°Cfor5-10minutes;stopincubationwhenedgesofcoloniesbegintopullawayfromtheplate. Aspiratethecollagenaseandaddappropriatevolume(3ml)ofconditionedmedia(CM)totheplate(seenotes). CollectcellsoffoftheplatebyscrapingandwashingwiththeCM,transfertoa15mltube. Aspiratematrigeloffof6wellplate(seematrigelaliquotingandplatingprocedure) Add2mlofCMperwellof6wellplate. Add5ulbFGF. Plate0.4ml/wellintoeachwelluntilthereisapproximately0.6mlremaining. Addtheremaining0.6mldropwisetoeachwelluntildone. Makesurethecellsareevenlydistributedacrosstheplate. Placegentlyintoincubator. Letsettleovernight. Notes: HighdensityMEFsforCMlastabout2weeksafterplating.Besuretocheckthemregularlyforcelldeath. bFGFisstableat4°Cforonemonth.
