3.12. MTT Assay

A 96-well plate seeded with A375 cells (5 × 10⁵/well) were incubated for 24 h for cell detachment, followed by removing the medium, washing with PBS once and treating with different concentrations of CEs or CNs (2, 5, 10, 15 and 20 µg/mL) for performing triplicate experiments. After incubation for 72 h and eventual removal of the medium, as well as washing with PBS, 200 µL of MTT solution (0.5 mg/mL) was added for reaction in the dark for 1 h and 200 μL of DMSO added to dissolve purple crystals for measurement of absorbance at 570 nm in an ELISA reader. Then, the cell viability was calculated based on the following Formula (2):

where, treatment = addition of CEs or CNs at different concentrations, blank = culture medium without cells, control = without sample treatment.