2.8. Tyrosinase activity assay

Tyrosinase activity was assayed as DOPA oxidase activity [24], and B16 cells were cultured in six-well plates at a density of 4 × 10⁵ cell/well with 100nM α-MSH. After 72 h, the cells were treated with various concentrations of PgAuNPs (1–100 μg/mL); after 72 h, the medium was removed and cells were washed with iced-cold PBS, and lysed with phosphate buffer (pH 6.9) containing 1% Triton X-100. The mixture was freeze-thawed by incubating at −80°C for 15 min and then kept at room temperature for 10 min. The samples were clarified by centrifugation at 12,000g for 15 min. After centrifugation, 10 μL of freshly prepared substrate solution (15mM l-DOPA in 50mM pH 7.1 sodium phosphate buffer) was added to 90 μL of lysate supernatant and incubated at 37°C for 1 h. The absorbance was then measured at 475 nm using an ELISA reader.