Potassium penicillin g

C2C12 cells were purchased from the Riken Cell Bank (Ibaraki, Japan). The C2C12 cells were maintained in growth medium (GM) composed of Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% foetal bovine serum (FBS; Sigma–Aldrich, St. Louis, MO) and antibiotics (100 U/mL potassium penicillin G and 0.1 mg/mL streptomycin (Invitrogen)). The cells were passaged before reaching confluence and used within ten passages. To induce differentiation, cells were cultured until confluence and differentiated in DMEM supplemented with 2% calf serum (Sigma–Aldrich) and antibiotics. Medium was exchanged every day. Mouse ESCs (E14Tg2a) were maintained on feeder-free gelatine-coated plates in LIF-supplemented medium: DMEM-High Glucose (DMEM-HG; Invitrogen, Carlsbad, CA) was supplemented with 10% FBS (Invitrogen), antibiotics (100 U/mL penicillin and 0.1 mg/mL streptomycin) (Invitrogen), 1 × GlutaMAX-1 (Invitrogen), 1% 2-mercaptoethanol (Bio-Rad), 1 × nonessential amino acids (Invitrogen), and 1 mM sodium pyruvate (Sigma-Aldrich).

Mouse melanoma cells, B16F1 (ATCC CRL-6323), were grown on 10 cm dishes cultured in Dulbecco's Modified Eagle Medium high glucose (DMEM, Invitrogen, Gaithersburg, MD, USA) to which was added 10% fetal bovine serum (Invitrogen), 0.1 µg/mL streptomycin sulfate, and 100 U/mL potassium penicillin G (Invitrogen).
Human umbilical vein endothelial cells (HUVEC) were provided as frozen cells after primary culture by the supplier (Kurabo, Osaka, Japan). HUVEC were utilized as a model for human endothelial cells, and cultured on 10 cm dishes in HuMedia-EB2 (Kurabo, Osaka, Japan) consisting of 2% fetal bovine serum, 10 ng/ml human epidermal growth factor (hEGF), 1.34 µg/mL hydrocortisone hemisuccinate, 50 µg/mL Gentamicin, 50 ng/mL Amphotericin B, 5 ng/mL hEGF-B, and 10 µg/mL heparin, all supplied by Kurabo. Cells were cultured in a humidified 5% CO₂ incubator at 37°C.