M16Medium(ProtocolobtainedfromKarenAusten-ReedfromSSTanLaboratory,AnatomyDepartment) Foroocytematurationandroutinecultureofmouseembryos,M16culturemediumisused.ThismediumisunabletomaintainitsownpHandmustthereforebyusedinconjuctionwithanincubatorbufferedwith5%CO2.TheCO2maintainstherequiredpHlevelofthemedium. AOR Penicillin/Streptomycin-useat1/100(TissueculturePen/Strep) Phenolred0.005 B NaHCO325.084.021.051 CNaPyruvate0.33110.00.018 DCaCl2-2H2O1.71147.20.126 BSA(BovineSerumAlbumin)4mg/ml Method Weighoutallofstock"A"(exceptPen/StrepandNalactate)intoameasuringcylinderandmakeupto90mlwith"Travenol"water.AddPen/StreptocylinderandthenNalactate(Note:theNalactateisquiteviscous-byheatingitupto~37oCpriortouseitcanbemoreeasilyandaccuratelyPipetted).Makeupto100mlwith"Travenol"waterandpourinto500mlbottle. Weighoutstock"B"(Phenolred,NaHCO3)componentsintoameasuringcylinder.Makeupto100mlwith"Travenol"andpourinto500mlbottle.Weighstock"C"(NaPyruvate)intomeasuringcylinders,makeupto100mlwith"Travenol"waterandpourinto500mlbottle.Weighstock"D"(CaCl2-2H2O)intomeasuringcylinder,makeupto100mlwith"Travenol"waterandpourinto500mlbottle.Addabout100mlof"Travenol"watertobringthetotalvolumeto500ml.Besurethatthecomponentsofeachstockhavedissolvedproperlybeforeaddingtothe500mlbottle.Alsobesuretoaddstocksintheiralphabeticalordertoavoidprecipitationofsomeingredients. Make50mlaliquotsofthissolutionandfreezethem.Useone50mlaliquotatatimebyaliquotingitintotubesof9mleach,thenrefrigerateuntiluse.Theosmolarityshouldbe288-292mosmol. Thismediumisusedformanipulationsthatareperformedonthemouseembryoswhilstoutoftheincubator.Sincethereisnot5%CO2presenttohelpmaintainthepH,thismediumcontainsHEPEStokeepthepHconstant. Ingredient%ofFinalVolume 1xDMEMwith20mMHEPESbuffer80.8% Penicillin/Streptomycin(5,000i.u/ml/5,000ug/ml)1% L-glutamine(200mM,100x)1% non-essentialaminoacid(100x)1% nucleosides(100x)1% b-mercaptoethanol(0.1M)0.2% Foetalcalfserum15% Thisrecipeismadeupfresheachweek(thesamestocksofingredientsmaybeused).AnalternativetoDMEMwithHEPESisM2,therecipeforwhichcanbefoundinBridgetteHogan"sBook,"ManipulatingtheMouseEmbryo".Compound mM Mol.Wt. g/500ml NaCl 94.66 58.45 2.766 KCl 4.78 74.557 0.178 KH2PO4 1.19 136.091 0.081 MgSO4-7H2O 1.19 246.5 0.146 MgSO4(anhydrous) 0.072 NaLactate 23.28 112.1 1.305(powderform)or1.69ml(60%syrup Glucose 5.56 179.86 0.5 Beforeuse,lightlygaswithCO2,thenadd1mlofFCS(foetalcalfserum)toa9mlaliquotofM16.Thensprinkle36mgofBSA(4mg/mlM16)ontopandallowtodissolve-donotshakeuporstir.ThenFilter-sterilizethismixusinga12mlsyringewithanacrodisc.
DMEMWITHHEPES
