成骨细胞培养.doc

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1、CalvarialosteoblastcultureTheoryOsteoblastsareisolatedfromthecalvariaeofmice・Thecalvariaiseasytoworkwith,easytoaccessandgivesplentyofcellsforexperimentalpurposes.HumanOBaredifficulttoisolateandveryslowgrowing,althoughhumanOBcelllinesexist,primarycellsa

2、remuchmoreinformativethancelllinesastheyareFormal'(manyOBcelllineshavenotbeenshowntoformboneandhavestrangestressresponses).Componentsrequired:1)Dulbecco9sModifiedEssentialMedium(DMEM)Toone500mlbottleofDMEMadd:10%foetalcalfserum(50ml)2mMl-glutamine(5ml)

3、100U/mlpenicillin,100gg/mlstreptomycin2)CollagenaseThisisa0.2%solutionandismadeupinHanksbalancedsaltsolution(HBSS).Tomake20mlofcollagenaserequires0.04gofcollagenasepowder(storedinthefreezer)・Oncethesolutionhasbeenmadeupitrequiresfiltersterilisingbefore

4、use.20mlofcollagenaseshouldbeseparatedbetween5tubesandstoredat-20°C(usewithin1month).3)SupplementedDMEM(notnecessaryforisolation)Thisshouldalwaysbemadeupfreshonthedayofmediumchange・Qa)Dexamethasone:AdddexatoafinalconcentrationofWMinsupplementedDMEM.Dex

5、amethasoneismadeupinethanol,useminimalvolumeforaddingtoyourculturemediumoryouwillkillyourcells(ie1/10,000dilution-stockat10'4M).b)p-glycerophosphate:For20misof0.4M,solutionyouneed1.7gp-glycerophosphatein20misPBS.Filtersterilizeandaliquotinto5mltubes,st

6、oreat-20(canbefreezethawedmultipletimes).c)Ascorbate:Tomake20mlof5mg/mlsolutionrequires0.1gofascorbatein20misofPBS.Filtersterilise.Aliquot500uLinto1.5mleppies,singleuse(donotfreezethaw).N.B.ascorbatedegradesveryrapidlysoonlyadditjustbeforeuse・Isolation

7、•Carefullyremovecalvarialbonefromanimal・Scrapeoffanyexcesstissueandcartilagefromaroundtheedge.•Placein5mltubeandwashwithPBS.(PBS洗儿遍？)•Coverwith0.25%Trypsin(体积要超过骨片，不要EDTA?)andincubateat37°Cfor10min(需要摇动吗？).•Removeanddiscardtrypsin;washwithDMEM/FCS(洗几遍,

8、需要摇动吗?).•Addenough(~600j.il)0.2%collagenase(几型胶原酶?)tocoverandincubateat37°Cfor30min(需要摇动吗？不同的鼠的年龄时间一致吗？)••Removecollagenasedigestanddiscardandreplacewithfreshsolutionincubateforafurther60minat37°C.(不需要摇动？，也是加600ul左右/每只鼠?)•Keepthefinaldi