SIGNIFICANCE - Mitochondrial and cellular reactive oxygen species (ROS) play important roles in both physiological and pathological processes. Different ROS, such as superoxide (O2(•-)), hydrogen peroxide, and peroxynitrite (ONOO(-)), stimulate distinct cell-signaling pathways and lead to diverse outcomes depending on their amount and subcellular localization. A variety of methods have been developed for ROS detection; however, many of these methods are not specific, do not allow subcellular localization, and can produce artifacts. In this review, we will critically analyze ROS detection and present advantages and the shortcomings of several available methods.
RECENT ADVANCES - In the past decade, a number of new fluorescent probes, electron-spin resonance approaches, and immunoassays have been developed. These new state-of-the-art methods provide improved selectivity and subcellular resolution for ROS detection.
CRITICAL ISSUES - Although new methods for HPLC superoxide detection, application of fluorescent boronate-containing probes, use of cell-targeted hydroxylamine spin probes, and immunospin trapping have been available for several years, there has been lack of translation of these into biomedical research, limiting their widespread use.
FUTURE DIRECTIONS - Additional studies to translate these new technologies from the test tube to physiological applications are needed and could lead to a wider application of these approaches to study mitochondrial and cellular ROS.