Measuring Cellular Superoxide Production

The specific probe hydroethidine (Thermo Fisher Scientific, Waltham, MA, United States, cat # D11347) was used to measure changes in cellular superoxide (O₂^•–) production (Sen and Hongpaisan, 2018 (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 (Zielonka and Kalyanaraman, 2010 (link)). Cultured cells, grown on glass slides, were incubated with hydroethidine (2 μg/ml) at room temperature for 15–20 min and then fixed with 4% paraformaldehyde. Increased O₂^•– production was qualitatively determined with a confocal microscope at 488 nm/>510 nm (excitation/emission).