Cell proliferation

Cell proliferation was assessed by the MTT colorimetric assay. At the end of each treatment, cells were collected, centrifuged, rinsed with PBS, stained with 1 mg/mL MTT solution, and incubated at 37°C/5% CO2 for three hours. Then, cells were centrifuged, treated with isopropanol, and incubated at room temperature for two hours. After incubation, absorbance was measured at 540 nm using a microplate reader (isopropanol was used as a blank). The viability rate for each experimental condition was calculated as the ratio of the average optical density of the treated cells to the average optical density of negative controls. Furthermore, the difference between the viability of the treated sample and the positive control was calculated, representing a direct index of the protective capacity of the product.