*5.8. Necrosis and Apoptosis Analysis*

HGEC were grown on gelatinized glass coverslips (12 mm) and then treated as it was described for item 5.7. After treatments, the percentage of necrotic and apoptotic cells were analyzed morphologically by fluorescence microscopy. For that, cells were stained with acridine orange/ethidium bromide (1:1, v/v) at a final concentration of 100 μg/mL [28]. In the analysis, it was considered that live cells have normal nuclei when presented with green chromatin and organized structures. Apoptotic cells contain fragmented or condensed chromatin (green or orange). Finally, necrotic cells have similar normal nuclei staining as

live cells, but with the chromatin in orange instead of green. The percentage of apoptotic and necrotic cells/field was obtained from photographs of 10 randomly selected fields. Cells were then counted, and the percentage of necrotic and apoptotic cells was estimated by considering the total number of cells/field as 100% (percentage of necrotic or apoptotic cells/field = (number of necrotic or apoptotic cells × 100)/total number of cells). Results were expressed as means ± standard deviation of the mean (SD). Percentages of prevention from necrosis and apoptosis were calculated by considering 100% of protection when these alterations were totally reversed.
