Cell culture
WEST
Murine C2C12 skeletal muscle cells (passage numbers 10–12) were seeded into 12-well plates and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (11966025, Gibco) containing 10% (vol/vol) fetal bovine serum (FBS; F9665, Sigma-Aldrich, St. Louis, MO), 1% (vol/vol) penicillin-streptomycin (15070–063, Gibco), 5 mM glucose (G7021, Sigma−Aldrich), 1 mM sodium pyruvate, and 1 mM GlutaMAX (35050–038, Gibco) under standard conditions (5% CO2, 100% humidity, 37℃). When the cells reached 90% confluence, the medium was switched to a differentiation medium consisting of DMEM supplemented with 2% calf serum (Bio West) and 1% nonessential amino acids (Invitrogen). This timing was designed Day 0. The differentiation medium was changed every 24 h. On Day 5 of differentiation, myotubes were changed to an amino acid and serum-free medium (D9800–13, US Biological, Salem, MA) (pH 7.3). The cells were incubated for 1 h without serum and amino acids prior to treatment. Myotubes were subsequently treated with a medium containing 10% human serum with placebo or MA before or after intervention for 24 h. The serum dose was determined from reflating previous studies, which ranged from 5% to 20% [