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.

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