HippocampalNeuronCultures(andmixedcorticalcultures)Submittedby:JeanSiao,Ph.D.BeginbytimingthepregnantmouseatE17-E19daysofgestation.Havereadythefollowing:1.ColdHank’sbalancedsaltsolution2.1xtrypsin(0.25%inHBSS)3.1xsoybeantrypsininhibitor(1mg/ml)---SBTI4.Neurobasalmediumwith:a.B27supplement(2mlper100mlmedium)b.L-glutamine(0.5mM)c.L-glutamate(25M)d.Gentamicin(10g/L)Thisiscalled“G3”medium.5.Poly-L-lysine-coateddishes,flasks,coverslips,etc.,thatyouwanttouse.6.Formixedcorticalcultures:DMEM+10%FBS+40ug/mlgentamicinOnthedayofdissectionandplating,placethetrypsin,SBTI,andG3mediuminthe37oCincubatortoequilibrate(temperatureandCO2).UncoverthetissuecultureplatesyouwantunderUVlighttodisinfectanddry.1.At,placetheinstrumentsinethanol（EtOH）andsomeHBSSintwopetridishesonice.Trytoburythemwithoutcontaminatingwithice.Puta15mltubeonicetoo.Ethanoldownthemicroscope.2.Sacrificethepregnantmousebycervicaldislocationoranotheracceptablemethod.OpentheaBDomenandremovethetwohornsoftheuteruswithethanol-sterilisedscissorsintoadishofHBSS.Takeoutthepupswhicharestillintheirplacentalsacs,andopenthesacswithouthurtingthepups’heads,whichareverysoft.Removetheumbilicalcord.Withbothfetusesandpups,usesterilisedscissorstocutofftheirheads.3.PlacetheheadsonthecoveroftheHBSSdishandethanolthemwell.4.Usethebluntforcepstoimmobilizetheheadstothedishthroughtheeyes.(Don’tsqueezeorpushthebrain!!)Usefinerforcepstopeelawaythescalpandthentheskull.Becarefulwiththefetusesbecausetheirtissuesareveryverysoft.Makesuretogetridofallofthesoftskull,elseyou’lldestroythebrainwhenyoutrytoremoveit.5.Usingthepointedendofthespatula,separatethebrainstemfromthespineandplacethebrainintotheotherdishofcoldHBSS.Makesuretokeepthatdishonice.Continuewiththerestoftheheads.6.Putthebrainsonthestageofthemicroscopeandadjustlightorfocussoyou’reconfortablewiththedissection.Usingthecurvedforceps(toimmobilisethecortexonthedish)andthedissectingscalpel,takeoffbothcorticesfollowingthecircleofWillis(redvessels).Trytouseacuttingmotionandgodirectlyupanddownthecircle.Puttherestofthebrainawayfordisposal.Continuewiththerestofthebrains.希望给大家帮助7.Removethemeningesfromthecorticeswithfineforceps.UseonepairtostABIlisethecortex,andtheothertogentlyliftthemeninges.Ifyou’reluckyit’llcomeawayinasheetstartingattheolfactorybulb:8.Turnthecortexaroundandfinishremovingthemeninges.Thehippocampusisconnectedattheconvexsidetothecortex,andattheconcavesidetomoremeninges.Getridofallofthemeningesusingfineforceps.9.Usefineforcepsto“cut”thehippocampusawayfromthecortexattheconvexside.Intheendyouwillhaveahalf-circlering.10.Putthehippocampiawaytooneside,makingsurethemeningesdon’tcontaminatethem.YoumightprefertohaveadishofcleanHBSSonicejustforthis.11.Ifyouwanttotakethecorticesformixedcorticalcultures,savetheremainderofthecortexandputintoanother15mltubethathasice-coldHBSSonice.Seebelow(step19)formixedcorticalcultures.12.Atthetissueculturehood,makesurethatthehippocampihavesettledtothebottomofthetubeandthenaspirateofftheHBSS.Add1mlofwarmedtrypsinper3brains(6hippocampi)andvortexthetube.Putintotheincubatorfor10-15minutes.13.AspirateoffthetrypsinandaddsamevolumeofwarmedSBTItohippocampi.Vortexwellandleaveatroomtemperature（RT）foracoupleofminutes.14.AspirateofftheSBTIandadd~2mlofwarmedG3mediumtohippocampi.15.Useasmoothly-fire-polishedPipettetotriturate.Whenyouseenochunksoftissue(afterabout15strokesyoushouldseeveryfew;youcandomaybeanother5strokesbutmorethanthatyou’llneedtostrainthroughastrainer—usethe70umonesbehindyou),stopandaddenoughmediumtothetubetoplate.Thismeansthatforthefirstcoupleoftimesyou’llneedtocountcellsbeforeplating,togetinyourheadtheapproximatenumberofcellsyoucandissect.Ifindithelpfultodilutethecellstothedesiredconcentrationandthenput100ulper12mmcoverslip—itsavesonmediumandthecellsarecongregatedonthecoverslip.16.Leavetheplateintheincubator.After3-5hoursofincubation,youhavetochangethemedium.Tiltthedishsothattheoldmediumrunsofftotheside,andaspiratethatoff.Carefulnottodisturbthesettlingcells.Addthefullamountofmediumperwellandreturntoincubator.17.After3-5daysinculturethemediummustbehalf-changedtoG2medium.ThisisthesameasG3,minustheglutamate.Tohalf-change,justaspirateoffcarefullyabouthalfoftheoldmediumandthenreplacethatvolumewithG2.18.Half-changethemediumevery3-5daysafterthatwithG2untilyou’rereadytousethem.19.AspiratetheHBSSoutofthetubecontainingthecortices.Add1mlper3-4brainsofwarmtrypsinandvortex.Incubateat37oCfor10min.20.Stopthetrypsinizationbyaspiratingoffthetrypsinandaddingequalamountofwarmmedium(DMEM+FBS).Vortex.IncubateatRTforacoupleofminutes.21.Aspirateoffthemediumandadd2-3mlwarmmediumtothecortices.Usingfirstaplainpasteurpipette,andthenafire-polishedpipette,triturateuntilalllargechunksoftissueisbrokenup.Thisshouldtakeabout20-35strokes.22.Filterthrougha70ummesh.Washthetubethatyoutrituratedinwith1mlmediumandaddthattothemesh.23.Ifyouwant,countcells.Iusuallyplateabout4brainsperT-125flaskthatwasprecoatedwithPLL.24.ChangethemediuminacoupleofdaystofreshDMEM+FBS+gentamicin.Afterthatchangemediumaboutevery5-7days.Thecultureshouldbereadyinabout10days.