Hydroethidine (Cat# 15200), a redox-sensitive probe, has been widely used to detect intracellular superoxide anion. It is a common assumption that the reaction between superoxide and hydroethidine results in the formation of a two-electron oxidized product, ethidium, which binds to DNA and leads to the enhancement of fluorescence (excitation, 500 - 530 nm; emission, 590 - 620 nm). However, the mechanism of hydroethidine oxidation by the superoxide anion still remains unclear. Hydroethidine operates effectively as a probe for the measurement of reactive oxygen species. The dye enters cells freely and is oxidized to ethidium bromide. The probe has been used extensively with NK cells and as a vital dye for identification of proliferation and hypoxic cells in tumors. Studies have been performed using neutrophils and endothelial cells as well as HL60 cells and macrophages. A major advantage of this probe is its ability to distinguish between superoxide and H₂O₂.