MacDermott 实验室原代神经细胞培养方法.doc

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1、MacDermottLabCellcultureprotocols(May1998;CristovaodeAlbuquerque)Contents1・Preparationofcoverslips2Massculture21.2-Agarose-collagenmicroislands2*1.3-uMacro,9-islands32・Dissection,plating,andmaintenaneeofcells32.1-Media3•IMDMforAstrocytes3•MEMforNeuron

2、s4•Neurobasal+B-2742・2・Corticalastrocytes47Dissectionandplating48Splittingoftheastrocytes59Astrocyteplating61.5Dorsalhornneurons6195.5Dissectionandplating6195.6Maintainingthecultures81.6Dissociateddorsalrootganglion(DRG)neurons9Dissectionandplating9Ma

3、intainingthecultures101.7Dorsalrootganglion(DRG)explants10Dissectionandplating103・Supplies&Suppliers114.Usefulreferences12Copyright©1998CristovaodeAlbuquerque1・Preparationofcoverslipsrk3bookmark4bookmark5bookmarMasscultureOurstandardmassculturesarepla

4、tedonastrocytes.Those,inturn,areplatedonglasscoverslipspre-coatedwithpoly・D-lysineandlaminin.Materials:#1coverslipscoverslipracksinawater-tightcontainer(wemadeours)poly-D-lysine(PDL)stocksolution(lmg/mlinddwater)lamininstocksolution(20(ig/mlinHank'sBS

5、S)35mmplasticculturedishesculturehoodequippedwithUVlampsterileddwaterProcedure:PlacethecoverslipsintheracksandleavethemintheculturehoodunderUVlightfor2hrs..Coatthecoverslipswith12.5

6、ig/mlPDL(5mlPDLin400mlsterileddwater)for2hrs.intheculturehood.Washthe

7、coverslipswithsterileddwaterfivetimes,intheculturehood.Placethecoverslipsinthesterile35mmdishesAdd0.4mllamininontopofthecoverslips.Waitfor45：thenaspiratetheexcesssolution1.2・Agarose-collagenmicroislandsThisprotocolisbasedonprotocolsbySegalandFurshpan.

8、Althoughthefollowingamacro-island?,approachhasallowedforgreaterneuronalsurvival,whilestillprovidingahighprobabilityofconnectionbetweenDRGanddorsalhorn(DH)autapsesorDH-DHconnectionscanonlybeobtainedwithhighprobabilityintheconventionalmicroislands.Mater

9、ials:#1coverslipscoatedwithPDLasabovetypeIIagaroseVitrogen100collagen,~3mg/ml35mmplasticculturedishesculturehoodequippedwithUVlampsterileddwateratomizerProcedure:Placecoverslipsin35mmdishesMeltagaroseinddwaterat0.2%,andplaceadroponthetopsurfac