20.109-Protocols/J1 Media

Culture Medium

Composition:

• DMEM (high glucose = 4.5 g/L), with supplements listed below
• 2 mM L-Glutamine
• 100 U/ml Penicillin/Streptomycin
• 0.1 mM β-ME
• 0.1 mM Non-Essential Amino Acid (NEAA)
• 10-15% Fetal Bovine Serum (Atlantic Biologic, Inc., Atlanta, GA)
  • ATCC recommends 15%, but in the past we have used 10% without problems.
• Leukemia Inhibitory Factor (LIF), 1000U/mL - LIF helps stem cells maintain their undifferentiated state

Protocol:

1. Mix all medium components together except LIF. For 0.5 L DMEM, use
   • 50 mL FBS
   • 5 mL NEAA
   • 5 mL P/S or P/S/G (check concentration of stock!)
   • 1 mL β-ME
2. Sterile filter using a 0.2 μm bottle-top filter.
3. Add the LIF in a sterile fashion. For, example
   • For 0.5 L of medium, pipet 1 mL of medium into a sterile eppendorf tube using a serological pipet.
   • Using a pipetman, add 50 μL LIF to the eppendorf tube. Submerge no more than necessary.
   • Use a serological pipet to return the 1 mL of concentrated LIF to the 0.5 L of medium.
   • The method above best keeps the pipetman barrel away from the sterile medium.

Pre-Transformation Medium

Culture medium above, less Pen/Strep. (Do add Glutamine.)

Freeze Medium

• 10% DMSO
• 10% extra FCS
• 80% standard culture medium