DissociatedCulturesofCerebellarNeurons

candooncounter(i.e.,innon-sterileconditions)useP8ratpups,orP5mousepupskeeponicewheneverpossIBLeforeachstep,doallpups,beforemovingtonextstepfill2tubeswithapprox.30mlHHGN(onefordissection;otherforcellprocessing--keepsterile);keeponicefillicetraysterilizetoolsbysoakinginEtOH:largeforceps,2fineforceps,curvedforceps,1smalland1largescissorsplaceTDn,DnBatroomtemptothaw

1. isolatecerebella(ÒCbÓ)

   cutoffheadintoplatewithHHGNholdnosewithlargeforcepscutwithscissorsthruskinandskull,fromsideofneck,totopofhead,across,andbacktoneckremoveflapofskin/skullfromfronttobackpinchoffcerebellumwithfineforceps,intoHHGN

2. removemeninges

   underdissectingscope,pullawayusingtwofineforcepsnotnecessarytoremoveentirely

3. chopeachcerebellumintoapprox4pieces(notnecessaryformouse,ifCbsmall)

4. Processing

   intissueculturehood;keepsteriletransfertissueto15mlpop-toptube(use25mlpipetsoopeningislarge)rinse3X2mlHHGNdigest15min,roomtemp,in5mlTDn(lowervolumeOKifdoingindividualCb,eg.formice)removesup,wash3X3mlHHGNadd5mlDnB;transferto50mltubetrituratewith5mlpipetuntilhomogeneous(20X)letsettle5minutestoremoveclumps;transfersuptosecondtube(optional:)re-triturateclumpswith5mlmoreDnB(morevigorously;sealpipettip,triturateapprox.10X)letsettle5minutes;transfer/poolsupwithfirstsupcentr500RPM,3min,R.T.resUSPpelletin10mlculturemedia(shouldresuspendeasily)countlive/deadcells:eg.:dilute1/10:65ulmedia(orHHGN)+25ul0.4%trypanblue+10ulcells[or,dilute3/4iflowconccells];count10ulexpectapprox50%livecellsdilutecellsinmedia,andseedplates/wells:24well:5X105livecells0.5ml35mm:2.5X1061.5ml60mm:5X1063ml100mm:1.5X1079mlnote,24w:shakeplatesvigorouslyleft-right,top-bottom,beforeplacingintoincubator,toevenlydistributecellsdonÕtneedtochangemediaafterseed(eg.,2h)growin5%CO2,at37Con1DIV(approx24h),addaraCto10uM:24well:10ul500uM35mm:30ul500uM[araC:-80CboxG5(HD)orE1]60mm:60ul500uMfeedwithnewmedia(containingaraC)on(3)-4DIV:1ml/3mlper60mm200ul/500ulper24well[candowhenreplacemediaaftertransfection]

5. DeathInduction

   on6DIV(havealsodoneon7DIV)wash2Xwith-KI24w:0.5ml35mm:1.5ml60mm:2mladd-KIor+KI

6. Reagents/Solutions
   1. HHGN

      500ml1XHBSS50ml10X[withoutCa,Mg;Gibco/BRL#14180-020]2.5mMHepespH7.3-7.51.25ml1Mglucose7ml2.5M(45%)NaHCO32ml1Mstoreat4C(approx2mo.)

   2. DNAaseBoehr-Mann#104159(2,000u/mg);bovinepancrease,gradeIIstock:4,000u/mlinH2O;sterilefilter,storeat-80C

   3. TDn250ulDNAase5mlHHGN50mgtrypsin[Worthington#3703]pHwith0.1NNaOHsterilefilternote,canmakeuplargeamount,aliquotto5mls,andstoreat-80C(approx1mo.)thawtoRTbeforeuse

   4. DnB500ulDNaseI10mlBME[Sigma#B1522]canmakeuplargeamount,aligoutto10mls,andstoreat-80CthawtoRTbeforeuse

   4. 3MKClmayneedtoautoclavetosterilize;tooviscoustofilter?

   5. BME+K500mlBME[5mMK]4.1ml3MKCl(therefore,24mMadditional,29mMKtotal)[unopenedBMEbottle:523mlonaverage]

   6. CalfSerum[Hyclone#A2151]needtoheatinactivatesterilefilter;aliquotto50mland10.5ml;storeat-20C

   7. CultureMedia(ÒCbCMedÓ)100mlBME+K10mlcalfserum1ml0.2Mglutamine[usefreshtube]1mlPenn-Strepwhenrefeed,addaraCto10uM:40ul25mMper100mlfinal[K]=26mM

   5. -KI100mlBME[5mMK]1ml0.2Mglutamine1mlPenn-Strep40ul25mMaraC[10uMfinal]

   6. +KIper10ml:10ml-KI25ul2mg/mlinsulin[5ug/mlfinal]67ul3MKCl[20mMadditional,final=25mM]sterilefilter(ifinsulinstockisunsterile)

   7. Notes

      usuallyuseP8Long-Evansrats;havealsousedP5-9successfully;P5/6ratsyieldedconsiderablyfewercells,andarelativelyhighernumberofglia;publishedprotocolsalsouseSprague-DawleyandWistarratsyieldofcellsisapprox1.5X107cellsperP8ratoriginalprotocolscallforfetalbovineserum;however,calfserumwasusedandseemssatisfactoryinsulinisusedforsurvival,ratherthanIGF-1ofref1,duetocostalthoughref1reportssurvivalinKClorIGF(inplaceofserum,at6DIV),mouseCbcellssurvivebetterwithbothpresent(forratCbcells,KClaloneallowedfullsurvival)withoutmediachange,inCbCmedia(serum+KCl),ratCbcellsliveonlyabout7-9days;with1change(4DIV),liveapprox12d

   8. References
      1. DÕMelloetal.(1993)PNAS90,p.10989

      2. Gallietal.(1995)J.Neurosci.15,p.1172