Measurement of cellular tyrosinase activity

Tyrosinase activity was estimated by measuring the rate of production of dopachrome from L-DOPA. B16 cells were treated as described above. Then, the cells were treated with α-MSH (100 nM) in the absence and presence of different concentrations of OEA (10, 30, and 50 μM) or kojic acid (200 μM) for an additional 72 h. After treatment, cells were washed twice with PBS and homogenized with 150 μl ice-cold PBS containing 1% (w/v) Triton X-100 (Sigma) and 0.1 mM phenylmethanesulfonylfluoride (PMSF) by freezing and thawing. The lysates were clarified by centrifugation at 13,000 r/min for 20 min, and then supernatants were collected. Afterward, the cellular extracts (90 μl) were transferred into a freshly prepared 10 μl L-DOPA solution (0.25% in PBS) in a well of a 96-well plate and incubated in the dark for 1 h at 37°C. After incubation, the generated dopachrome was monitored by an ELISA reader at the absorbance of 405 nm using the SpectraMax M5 microplate reader (Molecular Devices Corp., Sunnyvale, CA). Protein quantification of each lysate was performed using the BCA protein assay kit. For data analysis, the activity was calculated in units divided by the amount of protein measured.