2.4. MTS Cell Viability Assay

The cell viability MTS assay was performed according to the manufacturer’s instructions. Briefly, cells were seeded in a regular DMEM medium in 96-well plates (8 × 10³ cells/well) and allowed to adhere for 18 h, and then, the medium was removed. Next, the cells were washed twice with phosphate buffered saline (PBS) and finally treated with different canavanine concentrations (10–250 μM) in complete medium or arginine-free medium for 24, 48 and 72 h. MTS solution was added to each well, and the cells were further incubated for 1 h. The absorbance was measured using a Tecan SUNRISE XFluor4 plate reader at a wavelength of 490 nm. The percentage of viable cells after treatment was calculated by assuming 100% viability for the absorbance recorded for the control conditions (i.e., in the absence of canavanine).