原代神经细胞培养方法 Neuron Cell Culture

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1、1.Preparationofcoverslips1.1-MasscultureOurstandardmassculturesareplatedonastrocytes.Those,inturn,areplatedonglasscoverslipspre-coatedwithpoly-D-lysineandlaminin.Materials:.#1coverslips.coverslipracksinawater-tightcontainer(wemadeours).poly-D-lysine(PDL)stocksolution(1mg/mlinddwater).lami

2、ninstocksolution(20mg/mlinHank’sBSS).35mmplasticculturedishes.culturehoodequippedwithUVlamp.sterileddwaterProcedure:.PlacethecoverslipsintheracksandleavethemintheculturehoodunderUVlightfor2hrs..Coatthecoverslipswith12.5mg/mlPDL(5mlPDLin400mlsterileddwater)for2hrs.intheculturehood..Washthe

3、coverslipswithsterileddwaterfivetimes,intheculturehood..Placethecoverslipsinthesterile35mmdishes.Add0.4mllamininontopofthecoverslips.Waitfor45’,thenaspiratetheexcesssolution1.2-Agarose-collagenmicroislandsThisprotocolisbasedonprotocolsbySegalandFurshpan.Althoughthefollowing“macro-island”a

4、pproachhasallowedforgreaterneuronalsurvival,whilestillprovidingahighprobabilityofconnectionbetweenDRGanddorsalhorn(DH)autapsesorDH-DHconnectionscanonlybeobtainedwithhighprobabilityintheconventionalmicroislands.Materials:.#1coverslipscoatedwithPDLasabove.typeIIagarose.Vitrogen100collagen,~

5、3mg/ml.35mmplasticculturedishes.culturehoodequippedwithUVlamp.sterileddwater.atomizerProcedure:.Placecoverslipsin35mmdishes.Meltagaroseinddwaterat0.2%,andplaceadroponthetopsurfaceofeachcoverslip.Theheightofeachdropisdiminishedasmuchaspossiblebyremovingexcesssolutionwithapipettebeforetheag

6、arosegels..Allowcoatedcoverslipstoairdryovernightintheculturehoodatroomtemperaturetoformathinfilm.Foradequatedrying,thedishesmustbeuncovered..Spraythecollagenontothecoverslipswiththeatomizer.WeuseaglassperfumebottlewhichcanbeboughtatMacy’sinNewYork.Inourexperiencethatmakesbiggerdropletsth

7、antheFisherchromatographyatomizer,andismuchlessexpensive(andlooksbettertoo).Theatomizerisheldparalleltothebottomofthedishes,about25cmaboveandawayfromthem.Itisthenpumpedforcefullyafewtimes..Thecollagenislandscanbeexaminedwithaninvertedmicroscope.Theirsizeshouldbebetw