Measuring Cellular Tyrosinase Activity

The cellular tyrosinase activity were measured using tyrosinase Activity Assay Kit (Abcam, Cambridge, UK) and a slight modification of a previously reported method [40 (link)]. B16-F10 cells were seeded in 60 mm dishes (2 × 10⁵ cells/dish) for 24 h and treated with CFS (0.5 and 1% (v/v)) or arbutin (200 µM) for 40 h in the presence or absence of 200 nM α-MSH, harvested by trypsinization, sonicated in assay buffer (Abcam, Cambridge, UK), and centrifuged at 12,000 rpm for 20 min. The protein concentration was determined by the Pierce™ BCA Protein Assay Kit (Thermo Fischer Scientific). The reaction mixture consisting of 20 µg protein and 80 µL of 2 mg/mL L-DOPA (in 0.1 M sodium phosphate buffer, pH 6.8) was added to each well of a 96-well plate. After incubation at 37 °C for 1 h, the optical density at 492 nm was measured using a microplate reader.