Cells were seeded at a density of 2500/well of 96-well culture
plates and incubated for 24 h. The cells were treated with vehicle or
various concentrations of chalcone-21, followed by stimulated with
a-MSH (10 nM) or protoporphyrin IX (30 mM) for 3 days. Melanin
contents in cell-free culture medium were assayed by measuring
the absorbance at 405 nm using a microplate reader and were
calculated from a standard curve generated using synthetic
melanin. Kojic acid was used as a positive control.