UV-Induced Melanin Content Assay

Cellular melanin content was measured using a modified method by Jegal, et al. [75 (link)]. B16F10 cells were seeded at 5 × 10⁵ cells/mL in 24-well plates. Cells were then incubated for 24 h with 70 μM hdTIPs and 100 μg/mL kojic acid or arbutin as positive controls. After that, the cells in each well were washed and replaced by PBS. The UV stimulation process was performed by the exposure to UVA (314–400 nm, 3.0 W) and UVB (280–315 nm, 13.6 W) for 22 s. Then, PBS was replaced with media and further incubated for 24 h. The UV-stimulated cells were then trypsinized and centrifuged at 1500 rpm for 5 min. The cell pellet was then dissolved in 2 M NaOH at 60 °C for 1 h to solubilize the melanin. The melanin content was estimated by measuring the absorbance at 405 nm.
The melanin content was calculated with the following equation: $$\% Melanin content=\frac{\left(S\right)}{\left(C\right)}\times 100,$$
where, S is the OD₄₀₅ of sample and C represents the OD₄₀₅ of control. All experiments were performed in triplicate.