2.7. Cell Proliferation Assay

The cell proliferation effects of Iso A and Sec A on normal and melanoma cells were tested by MTT assay [14]. This method was based on the property that mitochondrial succinate dehydrogenase from living cells made MTT reduced to water-insoluble blue-violet crystal formazan and deposited in the cells, and dead cells without this function. Briefly, 1 × 10⁴ cells were seeded in a 96-well microplate. After 24 h, discarded the medium, cells were treated with various concentrations (10, 25, 50, and 100 μM) of Iso A and Sec A or vehicle control in a final volume of 100 μL culture medium. The experimental doses were accorded to our previous tests. After cultured within 37°C, 5% CO₂ incubator for 24 h, we replaced the medium with 100 μL fresh broth contained 0.5 mg/mL MTT. The plate was kept in a 37°C incubator for 2 h. We discarded the medium to add 100 μL DMSO dissolving the purple formazan crystals and gently shook the plate in darkness for 10 min. The optical absorbance values (A) were measured at 595 nm (BioTek, WA, USA). Cell viabilities were calculated according to the formula as follows: