4.6. Melanin Secretion and Content Assay

B16F10 cells (0.5 × 10⁵ cells/well) or NHEM cells (2 × 10⁵ cells/mL) were seeded into a 12-well plate and incubated for 24 h. The culture media were replaced with new, fresh media containing TSA (12.5–25 µM) and arbutin (1 mM). The α-MSH (100 nM) was subsequently used to stimulate the cells for 48 h. Afterwards, the secreted culture medium was measured by absorbance at 475 nm using a Spectra Max 250 microplate reader. Harvested cells were lysed by lysis buffer (50 mM Tris HCL pH 7.5, 20 mM β-glycerophosphate pH 7.5, 120 mM NaCl, and 2% NP-40) and centrifuged at 10,000× g for 3 min. Concentrated cell pellets were resuspended in 10% DMSO in 1 N NaOH and incubated in a heating block for 10 min at 60 °C. The melanin contents were measured at an absorbance of 405 nm using a Spectra Max 250 microplate reader. A melanin secretion and contents assay was conducted, as previously described [60].