2.9 Measurement of melanin content in B16F10 cells

Measurement of melanin content was carried out according to a previously described method with a slight modification (Karunarathne et al., 2019). B16F10 cells were cultured in 6-well plates at the density of 2 × 10⁵ cells/mL overnight before being treated with different concentrations of test samples for 48 h. After the treatments, the medium was discarded and the cells were washed twice with PBS and were collected, then 200 μL of RIPA lysis buffer was added to each well, and left standing in an ice bath for 40 min. The lysates were then clarified by centrifugation for 20 min at 4°C, 12,000 r/min. The protein content in the supernatants was measured using the Pierce BCA protein assay kit. Then the harvested cell pellets were photographed and dissolved in 100 μL of 1 M NaOH (containing 10% DMSO) at 100°C for 1 h. Melanin content was determined by measuring absorption at 405 nm. For the accurate calculation of melanin content, absorbance values were normalized to total protein absorbance values.