MediaUsed Toprepare100mlmedium Dulbecco"sModificationofEaglesMedium(DMEM) 1xwithoutL-glutaminewith4.5g/LGlucose.Storeat4 Cytosystems500mlCat.No.11.016.0500V Nonessentialaminoacids100x Cytosystems100mlCatNo.21-145-0100V.Storeat4 Penicillin-streptomycin(5,0001U/ml,5,000ug/ml) Cytosystems100mlCatNo.21-140-0100V.Storeat-20 FoetalCalfSerum(FCS) NeedstobetestedforABIlitytosupportgrowthofESCells.Serumshouldbestoredfrozenandheatinactivatedbeforeusebyheatingat56for30mins.Itmaybestoredat4followinginactiavation.Theheatinactivationremovescomplementactivitywhichalongwithnaturalheterophileantibodies,maybetoxicforEScells. 100xNucleosideStock Toprepare100ml(100x) Addto100mlTravenolwateranddissolvebywarmingto37.Filtersterilizeandaliquotwhilewarm.Storeat4formonths.Thenucleosideswillcomeoutofsolution.Warmto37beforeusetore-solubilize.Solutionseemstobestableat4C. Othersolutionsrequired. -1xDulbecco"sPhosphateBufferedSaltSolution(PBS)withoutcalciumandmagnesium. -Trypsin2.5%Cytosystems100ml21-159-0100V -Trypsin-EDTA(1:250)Cytosystems(0.05%Trypsin)100ml21-160-0100V Solutionstobemadeup PBS/EGTA(0.5mM) To100mlsofPBSadd1mlof0.05MstockEGTA(SigmaE4378)togiveafinalconcentrationof0.5mM. StockEGTAinH200.05Maddconc.NaOHtodissolve Trypsin0.25%. To20mlsCytosystemsTrypsin/EDTA0.05%,add1.6mls2.5%Trypsin 0.1%GelatininPBS To100mlsPBSadd0.1gmGelatinSigmaG-1890andautoclave 0.1M2-mercaptoethanol(E)SigmaM-6250 Add0.1mlE(14.4M)to14.3mlsPBSandfilterthrougha0.2uMACRODISC.Storeat-20foruptoonemonth. LIF(ifnofeedersareused) LIFESGROAMRAD(murineLifinPBS/BSAsolution)1mlampoule 107U/ml.Dilute1/100inDMEM10%FCS,aliquotinto10x10mltubes, storeat-20untiluse. 107U/mldilute1/100=105U/ml.(100xconc) Dilute1/100foruse=1000U/mlDMEM 80ml FCS 15ml Non-essentialaminoacids(100x) 1ml Pen/strep(5,0001U/ml,5000ug/ml) 1ml L-Glutamine200mM 1ml Nucleosidesstock(100x) 1ml BME0.1M 0.2ml Adenosine 80mg 3mM SigmaA4036 Guanosine 85mg 3mM SigmaG6264 Cytidine 73mg 3mM SigmaC4654 Uridine 73mg 3mM SigmaU3003 Thymidine 24mg 1mM SigmaT1895 Cytosystems 100ml 11.075.0100V 500ml 11.075.0500V
