Rpmi 1640 powder

Amino acid-free (AA−) medium was prepared by RPMI 1640 powder (R8999–04A, US Biological Life Science) and sodium phosphate dibasic (5.6 mM, the same concentration as commercially available RPMI 1640 medium, US Biological Life Science), supplemented with 10% (v/v) dialyzed FBS (Thermo Fisher Scientific). Amino acid-sufficient (AA+) medium was prepared by adding proper volumes of MEM amino acids solution (essential amino acids, EAA, 50×), MEM non-essential amino acids solution (NEAA, 100×), and 200 mM L-Gln (all from Sigma-Aldrich) to AA− medium to reach a final concentration of 1×EAA, 1×NEAA, and 2 mM Gln. The medium was supplemented with 10% (v/v) dialyzed FBS. Medium containing single amino acids (Ala, Leu, Gln, or Arg) or their combinations was prepared with AA– medium (prepared to the same concentrations present in the AA+ medium). All media were adjusted to pH7.5 and filter-sterilized (0.2 μm) before use.

Amino acid-deficient and amino acid-sufficient medium were homemade. Amino acid-free (AA–) medium was prepared using RPMI 1640 powder (R8999-04A, US Biological Life Science) and sodium phosphate dibasic (5.6 mM, the same concentration as commercially available RPMI 1640 medium, GIBCO) and supplemented with 10% dialyzed fetal bovine serum (FBS) (GIBCO). Amino acid-sufficient (AA+) medium was prepared by adding proper volumes of MEM amino acids solution (essential amino acids (EAA), 50×), MEM non-essential amino acids solution (NEAA, 100×) and 200 mM L-Gln (GIBCO). The medium was supplemented with 10% dialyzed FBS. Medium containing single amino acid (LEU or arginine (ARG)) was prepared with AA– medium (prepared to the same concentrations present in the AA+ medium), while medium deficient for single amino acid was prepared by adding all the EAAs or NEAAs to AA– medium, excluding the individual amino acids (same concentration as in AA+ medium). These media were adjusted to pH 7.5, filter-sterilized (0.2 mM) and supplemented with 10% dialyzed FBS (GIBCO) before use.