2.4. Melanin Content Analysis

B16F10 cells (1 × 10⁵ cells/well) were incubated in 24 well plates. In brief, B16F10 cells were treated with methyl gallate for 24 h. The cells were washed with PBS and then dissolved in 200 μl of 1N NaOH at 80°C for 2 h. The samples were centrifuged for 30 min at 12,000 rpm to collect the supernatant. The relative melanin content was determined by measuring the absorbance at 475 nm on a microtiter plate ELISA reader. The melanin content was determined using standard curves using synthetic melanin solutions (0∼200 μg/ml). Melanin production was calculated as the μg of melanin/μg of total proteins in a cell extract.