2.8. Determination of cellular melanin concentrations

Human melanoma cell line Mel 88.23 (SK‐MEL‐5) and murine melanoma cell line B16F10 were seeded at 0.5 × 10⁶ per well in 6‐well plate and cultured overnight in the presence of 1 mM L‐DOPA to stimulate melanin synthesis. Phenols were added to the cells at IC50 concentration and a twofold dilution range and cultured for 72 hr. Subsequently, cells were washed in PBS, 300 µl 1 M sodium hydroxide was added, and melanin was dissolved at 70°C for 120 min. Optical density of 200 µl of the dissolved cell solution, transferred to a 96‐well flat‐bottom microplate, was measured at 405 nm in a Versamax ELISA microplate reader. The melanin content of samples was calculated by a calibration curve of synthetic melanin (Sigma‐Aldrich) ranging from 0.49 to 500 µM melanin. The percentage inhibition of melanin synthesis in melanoma cells by phenol was calculated relative to untreated cells. Non‐pigmented cell lines HaCat and L cells served as negative controls for human and murine melanoma cells, respectively. A Bradford protein assay (Bio‐Rad, Utrecht, NL) was performed to relate the melanin content to the protein concentration, using a calibration curve of bovine serum albumin (BSA) in 1 M NaOH.