Quantification of melanin content

B16F10 cells (1×10⁵ cells/well) were plated and cultured in a 12-well plate overnight. After rinsing, cells were incubated with serum-free, phenol red-free DMEM for different periods of time. Cells and CM were collected by centrifugation for 400 × g, 10 min. CM were filtered through 0.22 μm filters. Cells were homogenized in 1 N sodium hydroxide (Duksan, Seoul, Korea) in 10% DMSO, and cell lysates were incubated in a 60°C water bath for 1 h. Cell lysates and CM were transferred to a 96-well flat-bottom plate and absorbance was measured at 490 nm using an ELISA reader to assess melanin content. Absorbance values were converted to amount melanin using a standard curve generated with synthetic melanin (Sigma-Aldrich). Melanin content was normalized to the total protein content of each sample, as follows. Total protein in cell lysates and CM was quantified by mixing 20 μL sample with bicinchoninic acid assay (BCA) reagent (Thermo Fisher Scientific, Waltham, MA, USA) and incubating at 37°C for 30 min. Absorbance at 562 nm was then measured using an ELISA reader. Normalized intracellular and secreted melanin content was calculated by dividing the measured melanin concentration by the total protein concentration.