Melanocyte and Melanoma Cell Culture Protocol

Adult human melanocytes (NHEM), derived from a healthy human subject, were purchased from Lonza (Walkersville, MD). Human melanoma cell lines (A2058, SK-MEL28, SK-MEL2, C8161 and 1205Lu) were purchased from ATCC (Manassas, VA) in a period between the year of 2014 to 2017 without further authentication. As described previously (34 (link)), frozen cells were newly thawed from low (3 (link)–10 (link)) passages. Mycoplasma was tested using PlasmoTest Mycoplasma detection kits (Invitrogen, Carlsbad, CA) and only negative cells were included in the experiments. Melanoma cells were maintained under a 5% CO₂ atmosphere in RPMI medium (Life technologies, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 Units/ml penicillin and 100 μg/ml of streptomycin. Melanocytes were cultured in 254 medium supplemented with Human melanocyte growth supplements (Thermo Scientific, Waltham, MA). Cells were seeded for 24 hours, and subsequently treated with sodium ascorbate (Sigma-Aldrich, St. Louis, MO). Media were changed daily to avoid the accumulation of unabsorbed ascorbate. All cells tested negative for mycoplasma by PCR.