Alpha melanocyte stimulating hormone

The kojic acid, trolox, l-ascorbic acid, l-4-hydroxyphenylalanine (l-tyrosine), l-3,4-dihydroxyphenylalanine (l-DOPA), alpha-melanocyte stimulating hormone (α-MSH), 3-isobutyl-1-methylxanthine (IBMX), 4-(1,1,3,3-tetramethylbutyl)phenylpolyethylene glycol (Triton™ X-100), phenylmethylsulfonyl fluoride (PMSF), 3-morpholinosydnonimine (SIN-1), dimethyl sulfoxide (DMSO), potassium hydrogen phosphate, potassium dihydrogen phosphate, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and mushroom tyrosinase were all purchased from Sigma-Aldrich (St. Louis, MO, USA).

The murine melanoma cell lines B16F10 and B16F1 (ATCC) were cultured in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. DMEM without phenol red was used for melanin measurements because phenol red, a pH indicator added to culture medium, can interfere with absorbance measurements. Cells were cultured in a humidified incubator in which 5% CO₂ was supplied and maintained at 37 °C. All cell culture supplies were purchased from ThermoFisher Scientific unless otherwise indicated. Alpha-melanocyte stimulating hormone (Sigma-Aldrich) was used to induce melanin synthesis in melanocytes. A stock solution of 0.5 mM of α-MSH was prepared in deionized water, and then diluted in a phenol red-free cell culture medium to final concentrations. Melanocytes were incubated for 24 hours at an initial concentration of approximately 1.8 × 10³ cells/cm². After 72 hours, the cell pellet and the culture medium were collected. The cell number was estimated from a cell counting using an automatic cell counter (Countess, Invitrogen).