4.3. MTT Cytotoxicity Assay

The cytotoxicity of melatonin was measured in the HT-29, SW48, and Caco-2 cells by the MTT colorimetric assay. The CRC cells were seeded at 5000 cells (in 100 μL culture medium) per well in 96-well plates and incubated for 24 h. Subsequently, the cells were treated with melatonin at various concentrations (0, 0.5, 1.0, 1.5, 2.0, and 2.5 mM). After an incubation for 72 h, the cells were incubated with 20 µL of MTT (5 mg/mL) solution for 4 h. Following medium removal, 100 µL of DMSO was added to each well and plates were gently shaken. The optical absorbance was measured at 570 nm, with 630 nm as reference wavelength, using Spectra Max3 Molecular Devices (San Jose, CA, USA). The absorbance of cells without treatment was regarded as 100% cell survival. Each experiment was repeated at least three times, beginning from the cell seeding to data analysis.