2.6. Cellular Tyrosinase Activity

Tyrosinase activity was determined by measuring the L-DOPA oxidation rate [29] with some modifications. B16F10 cells (5 × 10⁴ cells) were seeded overnight in 60-mm dishes and then treated with different final concentrations (2.5–10 μg/mL) of PME or IBMX. To inhibit p38 MAPK signaling, the cells were treated with 10 μM SB 203580 for 1 h prior to 10 μg/mL PME treatment. After 96 h incubation, the cells were washed with ice-cold Dulbecco's phosphate-buffered saline (DPBS) and lysed with PBS (50 mM, pH 6.8) containing 1% Triton X-100 and protease inhibitor cocktail (GenDEPOT Inc., Katy, TX, USA). The protein concentrations were then measured by BCA Protein Assay kit (Pierce Biotechnology), and the protein concentration in each cell lysate was equalized using lysis buffer. This was followed by the addition of 50 μL of each lysate and 100 μL of 5 mM L-DOPA to each well of a 96-well plate. The plate was kept at 37°C for 1 h in the dark and the absorbance was measured at 475 nm. Cells without PME or IBMX treatment served as the controls, and the relative activity of tyrosinase was expressed as (A₄₇₅ of treatment/A₄₇₅ of control) × 100%.