SF-AMEM

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)

73 mL 1X MEM (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 serum-free MEM adjusted to amphibian osmolarity.