2.7. Cellular Tyrosinase Activity Assay

Intracellular tyrosinase activity was determined by measuring the dopachrome formation of L-DOPA, as described in a previous study [32]. B16F10 melanoma cells were seeded at a density of 5 × 10³ cells/well in a 6-well plate. Cells were pretreated with 5 and 15 μM of brassinin for 1 h and further stimulated with 500 nM of α-MSH for 6 days. Cells were washed twice with cold PBS and dissolved with 130 μL of 50 mM sodium phosphate buffer (pH 6.5) containing 1% Triton X-100 and 0.1 mM PMSF. After freezing the cell lysates for 30 min at −80 °C, the supernatant was obtained by centrifugation at 12,000× g for 30 min at 4 °C. The 80 µL of the supernatant solution was placed in a 96-well plate, and then 20 μL of 2 mg/mL L-DOPA was added to each well. After incubation at 37 °C for 1 h, the absorbance was measured at 492 nm using a microplate reader. Intracellular tyrosinase activity was determined by normalizing the absorbance with total protein content and calculated as % of control.