1.Culturemedium:F12Kwithoutglutamine,3%FCS,100units/ml,100ug/mlstreptomycin(myocytemedium,alsoformixedculture);andF12Kwithglutamine,5-10%FCS,100units/mlpenicillin,100ug/mlstreptomycin(fibroblastmedium),onice.FCSshouldbescreenedforitsABIlitytosupportplatingandmyocytebeating. 2.Dissectiontools:1pairoflargescissors;2pairsfinescissors;2pairsofcoarseforceps;2pairsoffineforceps;soakingin95%EtOH. 3.SalineG-Ca2+andMg2+free. 4.Enzyme-0.125%trypsin+0.05%CollagenaseinsalineD.Theseconcentrationsworkwellfor6-7dayembryos.Olderembryosneed0.25%trypsinandahigherconcentrationofcollagenase.With6-dayandyoungerembryos,nocollagenaseisnecessary.Thawthesolutionandputintheincubatorat34or39oC..Need1ml/heart(formyocytes)or0.5ml/heart(forfibroblasts). 5.F12Kwithglutamine+20%FCS+1%penicillin/streptomycin.Samevolumeastrypsin-collagenase(~5ml),ina15mlconicaltubeandkeeponice. 6.Chamberdishes.Place1.5mlappropriatemediumoneachdishandincubateat34oC. 7.Preplateformyocyteculture.Add1mloffibroblastmediumtoa60mmdishandputinthe34or39oCincubator. 8.2x100mmsterilepetridishes-glassorplastic-onice. 9.Chickembryos,7-dayisoptimal,but6-or8-dayembryosarealsouseable.Need1-2embryosforeachchamberdish:oneperdishifnopreplating,2perdishwithpreplating.Eachheartyieldsabout105cells(myocytesplusfibroblasts). 10.15mlconicalcentrifugetubes. 1.Sprayeggs2xwith70%EtOH. 2.Wipetopsurfacewith95%EtOHthoroughly.Letdrycompletely. 3.Put1-2mlSalineGinoneofthepetridishestopoolheartsandkeeponiceuntiluse. 4.Removeembryostothesterile100mmpetridishwithoutsalineG.Itiseasiertograbtheembryosatfeet.Dissecteachembryooniceimmediatelyifyouareslow.Ifyouarefast,youcanremovealltheembryosfirstanddissectthemlater. 5.Beheadembryos.DissectandremovethewholeheartusingacleanpairoffinescissorsandforcepsandplaceinthedishwithsalineG.Heartscanbeidentifiedbasedonthepresenceofblood,beating,andassociatedbloodvessels. 6.Cuteachheartthroughtheventricletodrainanyremainingblood. 7.PipetoffsalineG.Reapply1-2mlsalineG,rinse,andpipetoff. 8.Repeatrinseonemoretime,oruntilsalineGlooksfairly"clean"(freeofblood).Morerinsesnecessaryformyocytes.LeavesomesalineGafterthefinalrinse,about0.2mlperheart. 9.Usingacleanpairoffinescissors,chopheartstosmallbutuniformsize-aboutthatofsandparticles.Becarefulnottochoptoolong,becausecellscanbedamaged. 10.Transfercellstoa15mlconicalcentrifugetube.Useasmallamountofsalinetorinsethedish.Repeattherinseifnecessary.Thetotalvolumeshouldbecloseto0.5mlperheart. 11.Trypsinization#1:mixtheenzymesolutionwithaPasteurpipet.Add0.5to1mlenzymetothetube.Mixbyshaking.Makesuretissuesarenotstickingtothesideofthetube.Placethetubeinthe34or39oincubatorforatotalof~5min.Shakeorvortexthetubeevery2-3min.Donotletsolutionsittoolongandbecometooturbid(willlosealotofcells).Keeptheremainingenzymesolutionwarm. 12.Removethetubefromtheincubatorandvortexat1/2speed.Lengthoftimevortexingdependsonthelengthoftrypsinization(shortvortexingforlongtrypsinization).Typically15-20sec.Shakepiecesoffthesideandletsettlefor1-2min. 13.Pipetoffsupernatantanddiscard. 14.Trypsinization#2:add0.5to1mlenzymetothesettledpieces.Vortexat1/2speedfor10-15sec.Incubatefor~5minasinstep11. 15.Removethetubefromtheincubatorandvortexatfullspeedfor10-15sec.Shakepiecesoffthesideandletsettlefor1-2min.SUSPensionneverlooksascloudyasafterthefirsttrypsinizationbecauseofthemuchsmallnumberofredbloodcells. 16.Transferthesupernatanttothetubewithcoldmediumcontaining20%FCS.Avoidclumps. 17.Repeatthetrypsinizationonthesettledmaterials.Theenzymesolutionshouldbedividedamongthenumberoftreatment.Formyocytes,repeat3-5times.Forfibroblasts,repeat1-2times.Accumulateallsupernatantinthesametubewith20%FCS. 18.Vortexhardattheendoflasttrypsinizationfor15sectotryandbreakupclumps.Pipetupanddowntobreakupclumpsevenmore. 19.Centrifugeintable-topcentrifugeonhighsetting(notquitemaxspeed)foratleast5min. 20.Removesupernatantanddiscard.Besurenottodisturbpellet. 21.Resuspendpelletin~1mlcoldmedium(for4hearts).Breakuppelletcompletelybypipeting. 22.Formyocytesonly,transferthesuspensiontothepreplatewithwarmfibroblastmedium.Incubatefor45-50min.Collectthesupernatantina15mlconicaltube. 23.Transferallsuspensiontoplates/dishes.Usuallyinnoculumsizeis~0.2mlperchamberdishor0.3mlper60mmdish.Forfibroblasts(withoutpreplating),eachchamberdishshouldcontain~6x104cells.Formyocytes,countcellsinthesupernatantafterpreplatingandplateat4x104cellsperchamberdish. 24.Changemedium2-3hrlaterforfibroblasts;24hrlaterformyoblasts.Useappropriatemedia.Feedcellsevery2-3days. 25.Cardiacmyocytesneed3-5daystospreadout.CulturingChickEmbryonicCardiacMyocytesandFibroblasts