Osteoblast Harvest(Adult Mice)

Material

• SacrificeAnimalsPlacemiceinabagandfillbagwithCO2-3-5minutes,alternativelyDryicemethod
• PourBetADIneoverhead
• SprayanimalswithEtoh
• CutSkinsagitallyfrombackofnecktowardsnose
• Removecalvariawithscissors-placeinPBSoniceinapetridish
• Scrapesofttissuesoffcalvariamacroscopicallyandmicroscopically(totalof2times)
• PlaceinPBS
• Mincewithscissorsintosmallpieces
• Wash2-3xinsuccessivedilutionsofBetadine:PBSandfinallywithPBS
• PlaceinDigestivemediainaconicaltube(75mlDMEM-alpha,0.150gDispaseII,0.075gCollagenaseA)
• Shakeat37ºfor10minutes-Discardfirstroundofdigestion-toomanyfibroblastsandunwantedcells-collectthesubsequentsupernatants
• Washandspin(1000rpm)3xwithPBStoremoveenzymes
• PlateinMediawithserum(10%)

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