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 &beta;-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
 * 2) *50 mL FBS
 * 3) *5 mL NEAA
 * 4) *5 mL P/S or P/S/G (check concentration of stock!)
 * 5) *1 mL &beta;-ME
 * 6) Sterile filter using a 0.2 &mu;m bottle-top filter.
 * 7) Add the LIF in a sterile fashion. For, example
 * 8) *For 0.5 L of medium, pipet 1 mL of medium into a sterile eppendorf tube using a serological pipet.
 * 9) *Using a pipetman, add 50 &mu;L LIF to the eppendorf tube. Submerge no more than necessary.
 * 10) *Use a serological pipet to return the 1 mL of concentrated LIF to the 0.5 L of medium.
 * 11) *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