2.7. In Vitro Cytotoxicity Assay

Cells were cultured in 96-well plates (7 × 10³ cells/well) in DMEM with 5% FBS. After 24 h of incubation, cells were treated with Ramosin peptide (0.5, 5, and 50 μM) and incubated for another 24 h. Cell viability was measured with the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma, reference: M5655) assay. Cells that were not treated were used as negative control and wells containing only medium and 5% FBS were left as blanks of the experiment. As a positive control, the cells were treated with hydrogen peroxide (H₂O₂) at 30 μM. The treatments with peptides at 0.5, 5, and 50 μM were carried out for 24 h at 37 °C and 5% CO₂. After this time, 10 μL of MTT (5 mg/mL) per well were added, and the plate was incubated at 37 °C and 5% CO₂ for 3 h. After this, 100 μL of isopropanol acid were added to each well, and the formazan crystals were dissolved with gentle stirring. The absorbance reading in the plate was performed in a MultiSkan GO at 570 nm. Finally, the cell viability percentage of each well was obtained, comparing treated cells and controls without treatment.