ProtocolforthePrimaryCultureofCorticalandHippocampalneurons

Solutionsandmediarequired:

• PolyD-lysine/lamininsolution-pdf
• DM/KY-pdf
• Optimem-pdf
• Neuronalgrowthmedium-pdf

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  Set-upforthedissection:

  Day1:

  AddpolyD-lysine/lamininsolutiontoculturedishes/coverslips.Swirltheplatetoensurethatthecoatingmixcoverstheentirebottomoftheplate.Leavethedishes/coverslipsinthe37oC/5%CO2incubatorovernight.

  Day2:

  1. Washthedishes/coverslipstwicewithsterilewater;removethefinalwashandleavethemliquid-freeintheincubator.
  2. MakeupDM/KY,sterilefilterandplaceonice
  3. MakeupthetrypsininhibitorsolutionandthepapainsolutionBUTDONOTaddpapainatthispoint;placesolutionsonice.
  4. Pourice-coldDM/KYsolutionintoseveralculturedishes:1largedishforthepupsand10cmdishesforthepupheads,fortheintactbrainsandforthedissectedcortices.Placedishesonice.
  5. Obtainpregnantrat

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  Dissectionofhippocampusandcortex:

  1. Sacrificetheratbyplacingaplasticdishcontainingdryiceinthecageandthenpouringwaterintothisplasticdish;coverthetopofthecagewithabag.
  2. Aftertheratfailstomovespontaneouslyorinresponsetopain(touchtheeyeandlookforareflex),incisealongtheabodmenandremovetheuterus.Placethepupsintothelargeculturedish.
  3. Removetheheadsofthepupsandplaceina10cmdish
  4. Foreachhead,removetheskinandcutalongthescalpinthemidlinewithfinescissors.Makeasimilarmidlinecutinthecalvarium.Deflectthecalvariumwithabluntspatulaandscoopthebrainintoanother10cmdishcontainingice-coldDM/KY.
  5. Dissectthecoritces:placethebrainventralsideup.Placethespatulainthemedialaspectoftheventralcortexandmidbrainandcutthecorticesoff.Discardthemidbrain.
  6. Dissectthehippocampusandcortex:placeacortexmedialsideupandplacethespatulaintothelateralventriclepushingforwardthroughthelateralaspectofthefrontalcortex.Extendthecutthroughthecortexinferiorlyandsuperiorlytoisolatetheposteriorhalfofcortexandhippocampus.Discardtheremainder.
  7. Dissectthehippocampusfromthecortex:placethespatulainthelateralventricleunderneaththehippocampus.Makecutssuperiorlyandinferiorlytofreetherespectiveendsofthehippocampus.Rolloutthehippocampuswiththespatulaandcutthehippocampusfromthecortexnearthedendate/entorrhinalcortexjunction.Placetheindividualcorticesorhippocampiin10cmdishcontainingice-coldDM/KY.
  8. Addthepapaintotheenzymesolutionbeforestrippingthemeninges(or30minbeforeanticipatedcompletionofdissection)andplacethepapainandtrypsininhibitorsolutionsina37oCwaterbath.
  9. Stripthemeningesandcutthetissueintosmall1mm3pieces.
  10. Sterilefilterthepapainandtrypsininhibitorsolutions.
  11. Transferallofthecortical/hippocampaltissuetoa15mlconicaltube.Useone15mltubeper5corticesdissected.OncethetissuehassettledremovetheextraDM/KYsolution.

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  Papaintreatment:

  1. Add5mloftheenzymesolutiontothedissectedtissueandincubateat37oCfor15min,mixingevery5min.
  2. After15min,removetheenzymesolutionandreplacewithasecond5mlofenzymesolution.Incubateforafurther15minwithmixingat5minintervals.
  3. After15min(totalof30min)removetheenzymesolutionandwashwithwarmedDM/KY3-4timesoruntilthetissuesolutionturnspink.

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  TrypsinInhibitortreatment:

  1.Add5mloftrypsininhibitor,mixthetissueandincubatefor5min.

  2.After5min,removethetrypsininhibitorandreplacewithfresh5mlaliquot

  3.Repeatatotalof3x/15mintrypsininhibitortreatment.Removeexcessandwastwicewith1mlofOptimem/glucose.

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  Trituration:

  1.Transfertissuetoa50mlconicaltubewith5mlOptimem/glucosesolution(3mlforhippocampus).

  2.Triturategentlywitha5mlPipetteuntilcloudy.Add3-5ml(1-2forhippocampus)ofoptimem/glucosesolutionandallowthetissuetosettleforafewminutes.Transfer3-5ml(1-2forhippocampus)ofcloudysupernatanttoasecondtube.

  3.Repeatstep2asmanytimesasnecessarytoobtainadequatenumbersofcells.Transfersupernatantfromsecondtubetoathirdtube,takingcaretoavoidanysmalltissuebitsthathavesettledintheinterim.

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  Platingcells:

  1.MixthecellsUSPensionandtransfer10ulaliquottoatubethatcontains10ulofDM/KYand10ulofTrypanblue.Mixthoroughlyandcountthenumberofcellsinthe16boxsquaresinthe2oppositecornersofthefield.Averagethe2countsorrecountifthe2numbersaredifferentbymorethan10%.Multipletheaverageby30,000togetthenumberofcellsperml.

  2.Dilutethecellswithoptimem/glucosesolutiontofinalcountof2.5-3millionpermlandplate2mlper60mmplate.After2hoursremovetheplatingmedia(optimem/glucose)andreplacewithgrowthmedia.