MEDIA – Mouse ES cell maintenance media：媒体–小鼠ES细胞维持液.doc

Serum Based mESC maintenance mediav DMEMUsually make 4L at a time. Add ingredients in the following order:IngredientsStocksWorking volumesDMEM (Gibco Powder w/oHepes, w/o NaHCo3)One liter packs(Cat# 12100-046)4 packsNaHCo33.025g/L12.1gCellgro water (Stir bar on)500ml6 bottlesHepes1M stock25mM (=1000mM/25mM= 40, 4L/40 =100ml)Penicillin/Streptomycin100X40mlCellgro water500ml2 bottles, bring solution up (stir bar off) to 4L by eyeFilter-sterilize in hood. Aliquot into eight 500ml bottles. Label with contents and date.Glutamine 100X1X = Add 5ml of Glutamine just before use.ORv DMEM-CellGro Usually reconstitute 500ml at a time. IngredientsStocksWorking volumesDMEM (MT15-017-CV1X500mlPenicillin/Streptomycin 100X5 mlGlutamine 100X5mlv IMDMUsually make 4L at a time. Filter-sterilize in hood. Aliquot into eight 500ml bottles. Label with contents and dateIngredientsStocksWorking volumesIMDM (Gibco Powder w/oHepes, w/o NaHCo3)One liter packs(Cat# 12200-069)4 packsNaHCo33.025g/L12.1gCellgro water (Stir bar on)500ml6 bottlesPenicillin/Streptomycin100X40mlCellgro water500ml2 bottles, bring solution up (stir bar off) to 4L by eyeFilter-sterilize in hood. Aliquot into eight 500ml bottles.Glutamine 100X1X = Add 5ml of Glutamine just before use.Home made IMDM:v Add in a bucket of 10 liters (reserved for cell culture only):v Powder 1X10liter cat# 12200-069 gibco v 8 liters of Cellgro water cat#25-055-CVv Add 30.2 grams of NaHCO3 (sigma cat# S-5761)v Stir at RT for at least 1 hrv Add Cellgro water for up to 10 litersv Stir againv Filter v Aliquot 750 ml in 1liter bottle and keep at 4C for 1-2 months protected from light.ORv IMDM Usually reconstitute 500ml at a time. IngredientsStocksWorking volumesIMDM (Cellgro #15-016-CV)1X500mlPenicillin/Streptomycin 100X5 mlGlutamine 100X5mlv DMEM-ES (Serum containing media)IngredientsStock Working volumes125mlHeat-inactivated ES-Qualified Gem Cell100%20% (Tested 10-20%)25mlLIF (batch dependent)100%0.5%-5% or 10ng/ml0.625-6.25mlMTG1.25gm/literDilute 100ul MTG stock with 900ul DMEM. Then add 12.6uL/100ml of DMEM-ES media15.75uLHome made DMEM1X1XMake up volume to 125mlv IMDM-ES (Serum containing media)IngredientsStock Working volumes125mlHeat-inactivated ES-Qualified Gem Cell100%20% (Tested 10-20%)25mlLIF (batch dependent)100%0.5%-5% or 10ng/ml0.625-6.25mlMTG1.25gm/literDilute 100ul MTG stock with 900ul DMEM. Then add 12.6uL/100ml of DMEM-ES media15.75uLHome made IMDM1X1XMake up volume to 125mlv DMEM-FCS or IMDM-FCS (Used to expand feeders)IngredientsStock Working volumes587 mlAtlas Serum100%15% 87mlMTG1.25gm/literDilute 26