AMEM (1%)
24 mL H2O (sterile; Merck)
1 mL antibiotic-antimycotic solution (AA solution; Sigma)
1 mL glutamine (Invitrogen)
1 mL insulin solution for amphibians
100 μL gentamicin (50 mg/mL; Sigma)
1 mL fetal calf serum, embryonic stem (ES) cell-qualified, heat-inactivated (Invitrogen) (see Note below)
72 mL MEM (1X; Invitrogen)
Prepare the medium in a sterile reagent bottle (e.g., a 75-cm2 tissue culture flask). Combine the reagents in the order listed. The resulting culture medium is MEM-adjusted to amphibian osmolarity plus serum for growth.
To heat-inactivate the complement system in the serum, defrost the stock bottle and incubate for 30 min at 56ºC. Cool the bottle to room temperature and store as 10-mL aliquots at −20ºC.
