Pitfalls of Reactive Oxygen Species (ROS) Measurements by Fluorescent Probes and Mitochondrial Superoxide Determination Using MitoSOX

Intracellular and mitochondrial superoxide formation is detected using phenanthrene-based dyes such as hydroethidine, mitochondria-targeted hydroethidine, or MitoSOX. HE and MitoSOX are redox probes, which undergo two-electron oxidation forming ethidium (E+) and Mito-ethidium (Mito-E+). The two-electron oxidation products derived from these probes exhibit the characteristic fluorescence that aids in fluorescence microscopy or flow cytometry or related techniques that are used to detect and determine superoxide (sometimes referred to as ROS, mitochondrial ROS, or mROS). This chapter briefly addresses the pitfalls of fluorescence-based techniques for detecting the intracellular superoxide.