2.4. MTT Viability Assay

Cell lines were treated either with five concentrations of gallic and ferulic acids, respectively, for 24 h, or in 70% ethanol as the control (suspended in DMEM alone). Adherent cells, as well as the L929 and Caco2 cells, were plated onto 96-well tissue culture plates (10⁵ cells/well) in complete medium. After treatments, proliferative cells were detected using the MTT assay, according to the ISO 10993-5 International Standard procedure (ISO 10993-5, 2009). The MTT substrate was prepared in DMEM, then added to cells in culture to attain a final concentration of 1 mg/mL, and then incubated for 2 h in the culture incubator at 37 °C with 5% CO₂. After incubation, the medium was removed by aspiration. Isopropanol (100 μL) was added to each well and formazan dye formation was evaluated by a multi-well scanning spectrophotometer at 540 nm. Suspension cells, of the U937 cell line, were plated at a concentration of 10⁵ cells/well onto 96-well culture plates in complete medium. After treatments, the cells were incubated with 0.5% MTT solution in PBS for 4 h at 37 °C, and then dissolved with 100 μL isopropanol in 0.04 N HCl. The plate was read at 540 nm and results were expressed as percentage of viable cells with respect to untreated controls (70% ethanol). The percentage of cell proliferation was calculated using the following formula: (absorbance value of treated sample/absorbance value of control) × 100 = % of cell viability.