資源描述：
《成骨細(xì)胞培養(yǎng).doc》由會員上傳分享，免費在線閱讀，更多相關(guān)內(nèi)容在工程資料-天天文庫。
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洗兒遍？)?Coverwith0.25%Trypsin(體積要超過骨片，不要EDTA?)andincubateat37°Cfor10min(需要搖動嗎？).?Removeanddiscardtrypsin;washwithDMEM/FCS(洗幾遍,
8、需要搖動嗎?).?Addenough(~600j.il)0.2%collagenase(幾型膠原酶?)tocoverandincubateat37°Cfor30min(需要搖動嗎？不同的鼠的年齡時間一致嗎？)??Removecollagenasedigestanddiscardandreplacewithfreshsolutionincubateforafurther60minat37°C.(不需要搖動？，也是加600ul左右/每只鼠?)?Keepthefinaldi
