2.2. Cytotoxicity Evaluation Using MTT Assay

To assess the vitamin C cytotoxicity in our study, the cell viability was evaluated using the MTT assay. Briefly, 5 × 10⁴ viable K-562 cells were placed into clear 96-well flat-bottom plates (Corning, New York, NY, USA) in DMEM high-glucose medium supplemented with 10% fetal calf serum and immediately after, different concentrations (0.1; 1; 10; 100; and 1000 μg/mL) of vitamin C were added to the cells. Following a 24 h incubation in a humidified atmosphere in a CO² incubator (5% CO₂, 37 °C), 10 μL/well of MTT (5 mg/mL) was added to the cells (both in the control and vitamin C and CQ treatments), which were incubated for an additional 4 h. After this time, 100 μL of a 10% sodium dodecyl sulfate (SDS) solution in deionized water was added to the cells and incubated overnight [23]. The absorbance was measured at 595 nm using a benchtop multimode SpectraMax i3 reader (Molecular Devices, San Jose, CA, USA).