3.5. Melanogenesis and Cell Viability

The effects on theophylline-stimulated melanogenesis and viability of B16 melanoma 4A5 cells were examined according to previously described protocols [21,22,23,24,25,26,27]. Briefly, murine B16 melanoma 4A5 cells were seeded into 48-well plates in DMEM. After 24 h of culture, a test compound and theophylline (1 mM) were added and incubated for 72 h. The melanin content and cell viability were then measured. IC₅₀ values were determined graphically.

Inhibition (%) was calculated using the following formula, where A and B indicate the optical density of the vehicle- and test compound-treated groups, respectively, and C indicates cell viability (%) (see below): Inhibition (%) = [(A − B)/A]/(C/100) × 100.