Serum Based mESC maintenance media

v  DMEM

Usually make 4L at a time. Add ingredients in the following order:

Ingredients / Stocks / Working volumes
DMEM (Gibco Powder w/o
Hepes, w/o NaHCo3) / One liter packs
(Cat# 12100-046) / 4 packs
NaHCo3 / 3.025g/L / 12.1g
Cellgro water (Stir bar on) / 500ml / 6 bottles
Hepes / 1M stock / 25mM (=1000mM/25mM= 40, 4L/40 =100ml)
Penicillin/Streptomycin / 100X / 40ml
Cellgro water / 500ml / 2 bottles, bring solution up (stir bar off) to 4L by eye
Filter-sterilize in hood. Aliquot into eight 500ml bottles. Label with contents and date.
Glutamine / 100X / 1X = Add 5ml of Glutamine just before use.

OR

v  DMEM-CellGro Usually reconstitute 500ml at a time.

Ingredients / Stocks / Working volumes
DMEM (MT15-017-CV / 1X / 500ml
Penicillin/Streptomycin / 100X / 5 ml
Glutamine / 100X / 5ml

v  IMDM

Usually make 4L at a time. Filter-sterilize in hood. Aliquot into eight 500ml bottles. Label with contents and date

Ingredients / Stocks / Working volumes
IMDM (Gibco Powder w/o
Hepes, w/o NaHCo3) / One liter packs
(Cat# 12200-069) / 4 packs
NaHCo3 / 3.025g/L / 12.1g
Cellgro water (Stir bar on) / 500ml / 6 bottles
Penicillin/Streptomycin / 100X / 40ml
Cellgro water / 500ml / 2 bottles, bring solution up (stir bar off) to 4L by eye
Filter-sterilize in hood. Aliquot into eight 500ml bottles.
Glutamine / 100X / 1X = 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-CV

v  Add 30.2 grams of NaHCO3 (sigma cat# S-5761)

v  Stir at RT for at least 1 hr

v  Add Cellgro water for up to 10 liters

v  Stir again

v  Filter

v  Aliquot 750 ml in 1liter bottle and keep at 4C for 1-2 months protected from light.

OR

v  IMDM Usually reconstitute 500ml at a time.

Ingredients / Stocks / Working volumes
IMDM (Cellgro #15-016-CV) / 1X / 500ml
Penicillin/Streptomycin / 100X / 5 ml
Glutamine / 100X / 5ml

v  DMEM-ES (Serum containing media)

Ingredients / Stock / Working volumes / 125ml
Heat-inactivated ES-Qualified Gem Cell / 100% / 20% (Tested 10-20%) / 25ml
LIF (batch dependent) / 100% / 0.5%-5% or 10ng/ml / 0.625-6.25ml
MTG / 1.25gm/liter / Dilute 100ul MTG stock with 900ul DMEM. Then add 12.6uL/100ml of DMEM-ES media / 15.75uL
Home made DMEM / 1X / 1X / Make up volume to 125ml

v  IMDM-ES (Serum containing media)

Ingredients / Stock / Working volumes / 125ml
Heat-inactivated ES-Qualified Gem Cell / 100% / 20% (Tested 10-20%) / 25ml
LIF (batch dependent) / 100% / 0.5%-5% or 10ng/ml / 0.625-6.25ml
MTG / 1.25gm/liter / Dilute 100ul MTG stock with 900ul DMEM. Then add 12.6uL/100ml of DMEM-ES media / 15.75uL
Home made IMDM / 1X / 1X / Make up volume to 125ml

v  DMEM-FCS or IMDM-FCS (Used to expand feeders)

Ingredients / Stock / Working volumes / 587 ml
Atlas Serum / 100% / 15% / 87ml
MTG / 1.25gm/liter / Dilute 26ul MTG stock with 2000ul DMEM / IMDM. Then add 3uL/ml to DMEM/ IMDM-FCS / 15.75uL
Homemade DMEM/IMDM / 1X / 1X / 500ml

Keep for 2 weeks or until pink