MTT cell proliferation assay

Melanoma A375, MV3, M14 cells, and other human cell lines including Hacat or HUVEC cells were seeded in 96-well plates with 5 × 10³/100 μL containing with 0.5, 1.0 or 4.0 μg/mL TanIIA for 24, 48, or 72 h culture respectively. The cells with dimethyl sulfoxide (0.1% DMSO, 0.0 μg/mL Tan II A) was used as negative control. Each treatment included six duplicate wells. To measure cell viability, 20 μl of MTT (5 μg/μL) (ATCC, Manassas, VA 20108, USA) was added to each well and incubated for 4 h at 37 °C in culture hood. The media was carefully removed and 150 μl MTT solvent was added to each well. The plate was covered with tinfoil and agitated on orbital shaker for 15 min. The absorbance (OD) at 490 nm (A) value was measured by micro-plate reader to determine the cells proliferation activity. The above experiment was repeated three times. The MTT reading level in negative control cell was used as 0% cell inhibition. The MTT reduction amount in Tan II A treated cells compared to negative control was normalized with negative control (0.1% DMSO, 0.0 μg/mL Tan II A) MTT level to calculate the inhibition percentage.