4.4. Intracellular Tyrosinase Activity Assay

Tyrosinase activity was determined as described previously, with slight modifications [29]. Briefly, Mel-Ab cells were seeded in 6-well plates and incubated with PDRN and Placentex^® for 4 days. Cells were then washed with ice-cold phosphate-buffered saline (PBS) and lysed with phosphate buffer (pH 6.8) containing 1% Triton X-100. The cells were then disrupted by a freeze/thaw cycle, and lysates were collected by centrifugation at 15,000 rpm for 10 min. After determination of protein levels, protein concentrations of all samples were equalized with lysis buffer. A total of 90 µL/sample was loaded used in each well of a 96-well plate, and 10 µL of 10 mM l-DOPA was added to all wells. Control wells contained 90 µL of lysis buffer and 10 µL of 10 mM l-DOPA. Following incubation at 37 °C, absorbance at 475 nm was measured every 10 min for at least 1 h using a microplate reader.