2.12. Tyrosinase activity assay

For the tyrosinase activity assay, SK-MEL31 cells were injected at a dose of 1 × 10⁵ cells per well in a 6-well plate. SK-MEL31 cells were incubated with minimum essential medium (MEM) 85%, FBS 15%, and 1% penicillin-streptomycin (10,000 U) at 37 °C in an incubator containing 5% CO₂ for 24 h. The medium was removed, the cells were washed with PBS, and tyrosinase was added and incubated for 72 h. The medium should be free of phenol red if possible, so that it does not affect the absorbance measurement. The medium may be treated with α-melanocyte-stimulating hormone (α-MSH) to promote melanin production. The medium was removed, cells were carefully cleaned with PBS and mixed with 0.1 M phosphate buffer and placed on ice for 1 h, waiting for the tyrosinase to be released from the melanosomes, and centrifuged at 13,000 rpm for 20 min at 4 °C. The supernatant was mixed with 0.2% L-DOPA solution, incubated at 37 °C for 30 min, and tyrosinase activity was measured at 490 nm using an ELISA reader.