CulturingChickEmbryonicMyoblasts/MyotubesandFibroblasts

Materials

Procedure

Materials

1.Culturemedium:Eagle"sMEMwith(exactly)10%horseserum,2%embryoextract,100units/mlpenicillin,100ug/mlstreptomycin,onice.

2.SalineG,onice.

3.SteriledistilledH2O.

4.Sterile15mlconicalcentrifugetubes.

5.Dissectiontools:2pairsofcoarseforceps,2pairsoffineforceps,1pairoflargescissors,2pairsoffinescissors,1pairofangularfinescissors.Soakin95%EtOHandairdryinhoodbeforeuse.

6.Eggcarton.

7.Ethanol(70%inaspraybottleand95%).

8.Twosetsofsterileglasspetridishes,sittingoniceinaplasticpan.

9.Twopetridisheswithgelatinsolution,preparedonday1.

10.Nitexfilter(20um),preassembledbytyingapieceofnitexaroundtheholderwithstringandautoclavedinaglasscan.

11.6mlplasticsyringe.

12.11-dayeggs(3embryosprovideenoughcellsfor10injectiondishes).

Procedure

1.Removegelatinsolutionfromtwopetridishes.Rinseplates2x,eachwith10mlsteriledistilledH2O.Makesureplatesarecompletelydryafterrinsing.

2.Add5mlmediumontoeachplate.Placein34^oCincubator.

3.Setupa"Preplate"byadding5mlmediumtoanuntreated100mmculturedish.Placein34^oCincubator.

4.Place2mlmediuminasterile15mlcentrifugetubeandletitsitonice.

5.Put~2mlSalineGinoneofthechilledsterilepetridish.

6.Sterilize6eggs:spraythetop(blunt)surface2xwith70%EtOH,letthemairdrybetweenthetwosprays.Thenwipewith95%EtOHusingKimwipe.Letdry.

7.Punchaholeontheshellwiththelargescissorsandcutaroundthetopsurface.Removetheembryobyholdingtheneckwithforceps.Onepairofforcepineachhandwillbeuseful.Collectupto7embryosonthechilledpetridishwithoutmedium.

8.Cutheadoffwithapairoffinescissors.Collectheadsinadisposablepetridishanddispose.

9.Remove,orpulloffskinfromthethighmuscle.Holdthedistalendofthelegwithoneforcep.Peelskinoffandfoldonthebody.

10.Dissecttheupperthighmuscleclosetothebody.Makesmallcutsonthemusclenearthejointswithapairoffinescissorsandpulloffmuslcewithfineforceps.Donotgoabovetheupperjointorlotsoffibroblastswillentertheculture.Ifdesirable,breastmusclescanbecollectedbyremovingskinfromthechestandpickingmusclesabovetheribcageandbetweentwowings.

11.CollectmuscleinthepetridishwithcoldsalineG.

12.Rinsemuscle2-3xwithicecoldsalineGtoremoveRBC.UseasterilePasteurpipet.Removeanyapparentpiecesofskinandbones.Remove~40%ofthefluidaftereachrinse.Thefluidshouldbeclear(notpink)attheendofthisstep.Thedishmaybeheldoutoftheiceforashortwhileifnecessary.

13.MincemuscleinasmallvolumeofsalineGuntiluniformpiecesareobtained.Keepthedishonice.

14.Transferthemincedmuscleto2mlmediuminasterilecentrifugetube(step4).UsesterilePasteurpipet.

15.Vortex30secatfullspeed.Allowpiecestosettleandthenpipetsupernatanttoanitexfilterapparatus.Keepthetubewithpiecesonice.

16.Filtersupernatantthrough20umnitexfilter.Gentlyapplynegativepressurewitha6mlplasticsyringe.Holdthefilteratananglesothatfiltratecanflowalongthesideofthetube.Keepthecollectingtubeonice.

17.Add2mlcoldmediumtosettledpieces.Themediumcanbeusedtorinseoutthepetridishbeforeaddingtothetube.

18.Vortex1/2speedfor15sec.

19.Filtersupernatantthroughsamefilter.

20.Get"Preplate"fromtheincubator.Spreadthefiltrateontheplate.

21.Incubate25mininthe34^oCincubator.

22.Afterincubation,dividesupernatantintotwohalvesandaddthemtothetwogelatin-coatedplates.Putinthe34^oCincubatorfor24hr.

23.Preparecollagen-coatedplates/dishesasdescribedseparatelylater.Need16plates.

Materials

1.SalineG,in34^oCwaterbath.

2.Soybeantrypsininhibitorsolid.

3.Medium,asforday2,onice.

4.3xcrystallizedtrypsininsalineG,onice.

5.Millex-GVfilterandsyringe.

6.15mlconicalcentrifugetubes.

Procedure

1.Finishthepreparationofcollagen-coateddishesasdescribedlater.

2.Measure1-2mgsoybeantrypsininhibitor.Dissolveincoldmediumataconcentrationof0.5mg/ml.

3.SterilizetrypsininhibitorwiththeMillex-GVfilter.Filterinto15mlconicalcentrifugetubes.Eachtubecontains0.5mlandwillbeusedforonegelatin-coatedplate.Keepthemonice.

4.Checktheprimarycultureongelatin-coateddishes.Thereshouldbemanysinglecellsandlittlefusion.Aspirateoffmedium.

5.Rinsedisheswith10mlwarmsalineG.AddthesalineGslowlyattheedge.

6.RemovesalineGcompletely.Add5mlcoldtrypsintoeachdish.Letitsitatroomtemperatureandswirloccationally.

7.Checkcellsunderthemicroscope.Within2-5min,about50%ofmyoblastsshoulddetachandtheother50%becomeround,whilemostfibroblastsshouldremainattachedtothedish.

8.Washmyoblastsoffthedishbytiltingdish45oanddripmediumneartheuppersideofthedishwithaPasteurpipet.Rotatedish90oandrepeattheprocess.

9.Transfersolutiontothetubewithcoldsoybeantrypsininhibitor.Mixbypipeting.

10.Countcellswithhemocytometer.Keepthetubeonice.Cellnumberperml=numberin25smallsquaresx104.

11.Diluteifnecessary.Platecellsontocollagen-coateddishes.

Numberperdish=4x103;8x103;1.2x104;1.6x104.

Performserialdilutionwithcoldmediumifnecessary.MixwellandkeepthesUSPensioncold.Thevolumeaddedtoeachchamberdishshouldbe0.1-0.3ml.Mixthesuspensionwellanduseglass1or2mlpipettoremovecells.Innoculatequicklytoavoidcellssettling.

12.Swirldishtospreadcellsoutandplacein39oCincubator.

Feedwith2mlfreshmediumperchamberdish.

1.Examineplates.

2.ApplyaraCifthereisapparentfusion,i.e.presenceofmyotubeswith~10nucles.

3.Afterwardsreplacemediumeverythirdday(noarac).

AraCStock(1mM)

1.Add1mgaracto2.5mldistilledH2O.

2.SterilefilterthroughMillex-GVfilter.

3.Storeinfreezer(-20^oC).

4.Useat5x10-6Minregularskeletalmusclemedium(100ularacin20mlmediumorpipet10uldirectlyontochamberdishwith2mlmedium).

GelatinStock(100ug/ml)

1.Add10mggelatinto100mldistilledH2Oinamediumbottle.

2.Sterilzebyautoclavingfor20min(slowexhaust),mixthoroughly.

3.Storeat4^oC(stableformonths).

CoatingChamberDishwithCollagen

Materials

1.Solublecalfskincollagen(CooperLS01660),diluted1:100withsteriledistilledH2O(1ml+100ml)andstoredat4^oC.

2.3.76MsterileNaCl(filteredwith0.22umfilter).

3.Chamberdishes,sterile.

Procedure(performinsterilehood)

1.Pipetdilutedcollagensolutionintoasterileflask(need1.5mlcollagenperchamberdish).

2.Add40ul3.76MsterileNaClpermlcollagen.Mixgently.

3.Platecollagensolutionontochamberdishes.

4.LetsitovernightunderUVlightinthesterilehoodwithlidspartiallyoff.

5.Aspirateoffcollagensolution.

6.Rinse2xwithsteriledistilledH2O.RemoveallH2O.Donotscratchthecoatedsurfaceofthedish.

7.Pipet1.5mldesiredmediumontodishesandputinthe39^oCincubator.

CHICKSKELETALMUSCLEFIBROBLASTS

Materials

1.F12Kwithglutamine,10%FCS,1%penicillin/streptomycin.

2.100mmtissueculturedishwith5mlmedium,incubatedat34^oC.

3.Sterile15mlconicaltubewith0.5mlsoybeantrypsininhibitor.Preparedinthesamewayasformyotubeculture,Day3.>

4.3xcrystylizedtrypsininsalineG,thawandkeeponice.

5.STE,in34^oCwaterbath.

6."Preplate"frommyotubeculture,Day2step22.

Procedure

1.Rinsepreplatewith10mlSTE.

2.Apply5mlcold3xcrystalizedtrypsin.Swirlandwaituntil50%ofcellshavedetached.

3.Pipettrypsinontocellstoremovemorecells.

4.Pipetthesuspensionintothetubewith0.5mlsoybeantrypsininhibitor.

5.Plateontothedishwith5mlwarmF12Kmedium.