Melanin

Melanin content was measured as described previously (Chung et al., 2019 (link)) with some modifications. B16F0 cells (100,000) were seeded in a 6-well plate and incubated at 37 °C/5% CO₂ overnight. On the following day, cells were treated with the extracts for 3 days in the presence of 0.1 μM α-MSH (Sigma-Aldrich, Sydney, Australia). Then, cells were collected, washed, and resuspended in 150 μL of 1N NaOH, and the cell suspension incubated at 60 °C for 1 h. Melanin (Sigma-Aldrich, Sydney, Australia) standards were prepared in 1N NaOH at 80 μg/mL, 40 μg/mL, 20 μg/mL, 10 μg/mL 5 μg/mL and 2.5 μg/mL of Melanin standard solutions using 1N NaOH. Then 100 μL of each Melanin standard was transferred to a 96-well plate in triplicate to obtain a standard curve. The cell extracts were diluted 1/2 using 1N NaOH, and 100 μL of the diluted cell extracts were transferred to appropriate wells of a 96-well plate in duplicate. The 96-well plate was mixed in the SpectraMax M3 plate reader (Molecular Devices, California, United States) for 30 s, and the absorbance was measured at 405 nm. The Melanin amount of each sample was calculated using the Melanin standard curve. Lowry protein estimation was used to determine the amounts of total protein in each cell sample. Therefore, the μg of Melanin per mg of total protein was calculated for each cell sample (Chung et al., 2019 (link)).