Measuring Cellular Superoxide Production

The specific probe hydroethidine (Thermo Fisher Scientific, Grand Island, NY, USA) was used to measure changes in cellular superoxide (O₂•−) production[59 (link)]. The reaction between O₂•− and non-fluorescent hydroethidine generates a highly specific red fluorescent product 2-hydroxyethidium. In biological systems, another red fluorescent product ethidium is also formed, usually at a much higher concentration than 2-hydroxyethidium[60 (link)]. Cultured cells, grown on glass slides (4 independent experiments per group), were incubated with hydroethidine (2 μg/ml) at room temperature for 15–20 min and then fixed with 4% paraformaldehyde. Increased O₂•− production was quantified at 488 nm/ > 510 nm (excitation/emission). For confocal microscopy studies, the cultured cells were fixed with 4% paraformaldehyde at room temperature for 15–20 min and stored in phosphate buffer saline at 4