1. -embryosaredissectedfromtimed-pregnantmicefrom10.5-11.5d.p.c.
2. -limbbudsaremicrodissectedandplacedinholdingmedium(L15mediumsupplementedwith1xMITO+serumextender)(allmediaissupplementedwith100Upenicillin/mland0.1mgstreptomycin/ml)
3. -limbsareallowedtositinholdingmediumanywherefrom20minto2hours
4. -0.2mlofserum-freeexplantmedia(DMEM-F12mediumsupplementedwith5xMITO+withorwithout10%fetalbovineserum)isaddedtoeachwellofasix-welltissuecultureplatecontainingCyclopore(polyethyleneterphthalate;FalconLabware)inserts
5. -limbsaretransferredfromtheholdingmediaontothesurfaceoftheCycloporemembranes(fortransfer,theendofa200祃Pipettetipisremovedwithasterilerazorblade(toenlargethehole),andlimbsaretakenupin5–10祃ofholdingmedia)
6. -limbsaregrownonthetissuecultureinsertsina37oCincubatorunderanatmosphereof5%CO2
7. -explantmediaisreplacedevery48hours

Limbscanbesuccessfullygrownfor3–4days

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