Background: Nearly ten years ago, we demonstrated that superoxide radical anion (O2⋅¯) reacts with the hydroethidine dye (HE, also known as dihydroethidium, DHE) to form a diagnostic marker product, 2-hydroxyethidium (2-OH-E(+)). This particular product is not derived from reacting HE with other biologically relevant oxidants (hydrogen peroxide, hydroxyl radical, or peroxynitrite). This discovery negated the longstanding view that O2⋅¯ reacts with HE to form the other oxidation product, ethidium (E(+)). It became clear that due to the overlapping fluorescence spectra of E(+) and 2-OH-E(+), fluorescence-based techniques using the "red fluorescence" are not suitable for detecting and measuring O2⋅¯ in cells using HE or other structurally analogous fluorogenic probes (MitoSOX(TM) Red or hydropropidine). However, using HPLC-based assays, 2-OH-E(+) and analogous hydroxylated products can be easily detected and quickly separated from other oxidation products.

Scope of review: The principles discussed in this chapter are generally applicable in free radical biology and medicine, redox biology, and clinical and translational research. The assays developed here could be used to discover new and targeted inhibitors for various superoxide-producing enzymes, including NADPH oxidase (NOX) isoforms.

Major conclusions: HPLC-based approaches using site-specific HE-based fluorogenic probes are eminently suitable for monitoring O2⋅¯ in intra- and extracellular compartments and in mitochondria. The use of fluorescence-microscopic methods should be avoided because of spectral overlapping characteristics of O2⋅¯-derived marker product and other, non-specific oxidized fluorescent products formed from these probes.

General significance: Methodologies and site-specific fluorescent probes described in this review can be suitably employed to delineate oxy radical dependent mechanisms in cells under physiological and pathological conditions. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.