4.3. Melanin Content and Tyrosinase Activity Assay in B16F10 Cells

The melanin content and tyrosinase activity of B16F10 cells were measured according to a method described in previous studies [34] and using an enzyme-linked immunosorbent assay (ELISA) reader (Tecan, Grodig, Austria) at 405 nm. Brifely, the B16F10 cells were cultured in six-well culture plates and incubated. The cells were treated with a medium containing α-MSH and various concentrations of sesamol for 48 h. NaOH (2N) was added to each well to lyse the cells, and the cells were then centrifuged. The amounts of melanin in the supernatant were spectrophotometrically measured at 405 nm

In the tyrosinase activity assay, the B16F10 melanoma cells were plated in a 24-well plate and treated with a medium containing α-MSH and various concentrations of sesamol for 48 h. The medium was removed and then 1% Triton X-100 mixed in 100 mM phosphate buffered saline was added. The mixture was frozen at −80 °C and thawed at room temperature, and then centrifuged. A freshly prepared substrate (15 mM l-DOPA) was then added to the supernatant and incubated. The absorbance of each well was subsequently read.