2.3.4. Cellular Tyrosinase Activity Assay in B16F10 Cells

B16F10 cells were seeded at a density of 3 × 10³/well in 6-well plates for 24 h at 37 °C in an atmosphere of 95% air/5% CO₂. Each test agent was dissolved in DMSO at a final concentration up to 20 µM and pretreated with the indicated concentration of each compound; then, α-MSH (5 µM) and IBMX (200 µM) were added to the medium for 48 h. After treatment, the cells were washed with ice-PBS and lysed by the addition of 50 mM phosphate buffer containing 1% Triton X-100 and 1% PMSF at −80 °C for 1 h. The lysates were clarified by centrifugation at 5600× g for 10 min at 4 °C; then, 80 µL of the cell lysate was added to 20 µL of l-DOPA (2 mg/mL in distilled water). The mixture was incubated for 10 min at 37 °C, and the absorbance of the reaction mixture at 492 nm was recorded using a microplate reader (Tecan, Männedorf, Switzerland). Kojic acid was used as the positive control.