2.7.2. Determination of Melanin Levels

B16F10 cells (1 × 10⁵ cells/well) were seeded in 12-well plates and cultured for 24 h. The medium was then replaced with compounds at nontoxic concentrations that were selected on the basis of the MTS assay and incubated for another 48 h. After the treatments, the cells were detached using TrypLE (1×), and cell pellets were washed in PBS. Cell pellets were visually observed for lightening of the pigment. After aspiration, 250 μL of 1N NaOH was added and heated to 70 °C to solubilize melanin. The aliquots were then transferred to a 96-well plate and absorbance was read at 475 nm using a microplate reader. The absorbance was normalized to total protein content and was expressed as % of control.