Ccp fbs br 500

HeLa cells were cultured in minimum essential medium (MEM, Gibco, 11095072, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS, CCP-FBS-BR-500, Cosmo Bio, Tokyo, Japan) and 1% penicillin-streptomycin (10,000 U/mL, Thermo Fisher Scientific, Waltham, MA, USA) for 3 days. The adherence of HEK-293 cells to the Petri dish was relatively weak; hence, the glass Petri dish was coated with fibronectin before seeding cells. For HEK-293 cells, glass Petri dishes were first coated with fibronectin and placed in humidified atmosphere incubator with 5% CO₂ at 37 °C. After coating the dish with fibronectin, HEK-293 cells were cultured. Both HEK-293 and SAOS-2 cells were cultured using high glucose DMEM (FUJIFILM Wako Pure Chemical Co., Ltd., Tokyo, Japan) with 10% FBS and 1% penicillin–streptomycin (PS).

The B16F10 murine melanoma cell line and A375 human melanoma cell line were obtained from the American Type Culture Collection. B16F10 cells and A375 cells were cultured in Dulbecco’s Modified Eagle’s Medium (D5796; Sigma Chemicals, St. Lousi, MO, USA) supplemented with 1% Modified Eagle’s Medium Non-Essential Amino Acids (11140050; Life Technologies, Carlsbad, CA, USA), 1% HEPES (15630080; Life Technologies), 1% sodium pyruvate (11360070; Life Technologies), 5% fetal bovine serum (CCP-FBS-BR-500; Cosmo Bio, Tokyo, Japan), 2 mM L-glutamine, 100 units/mL penicillin, and 10 µg/mL streptomycin (10378016; Life Technologies).