Dihydroethidium

Dihydroethidium (DHE) is a cell-permeable fluorescent probe widely used to detect intracellular reactive oxygen species, especially the superoxide anion. Inside cells, DHE is oxidized to fluorescent products. The reaction with superoxide yields 2-hydroxyethidium, a red-fluorescent compound that intercalates into DNA and can be observed by fluorescence microscopy or flow cytometry. A competing oxidation pathway, however, produces ethidium, which is also fluorescent but not specific for superoxide. Because both products fluoresce in a similar spectral range, measurements based on total red fluorescence can overestimate superoxide unless specific methods are used to distinguish them, such as high-performance liquid chromatography (HPLC) or spectral imaging with unmixing.

DHE is commonly used to monitor inducible or basal intracellular superoxide production in cells and tissues, under various stimuli, inflammatory conditions, ischemia-reperfusion models, and in mitochondrial studies. The dye is typically used at micromolar concentrations and requires careful handling to minimize photo-oxidation; samples should be kept away from light, and appropriate negative controls should be included.

Limitations: DHE can be oxidized by other reactive species, including peroxynitrite and hydroxyl radicals, leading to non-specific fluorescence. Photobleaching and artifacts can further complicate interpretation. Consequently, DHE provides semi-quantitative readouts of oxidative stress and is often used for relative comparisons rather than absolute measurements. For robust conclusions, complementary assays or chromatographic separation are recommended to distinguish 2-hydroxyethidium from ethidium.