Methods to detect hydrogen peroxide in living cells: Possibilities and pitfalls

Intracellular generation of reactive oxygen species (ROS) is an inescapable consequence of aerobic metabolism. Although some of these oxygen-derived metabolites are well-documented mediators of cell and tissue damage, others have been shown to be crucial for cell survival and homeostasis. One ROS that has been identified as a major second messenger in redox signaling is hydrogen peroxide (H2O2). This small, membrane-permeable oxidant is produced transiently in physiological (nontoxic) amounts by a variety of different enzymes residing within different subcellular compartments and organelles. There is an accumulating literature demonstrating that the reversible, H2O2-mediated oxidation of different signaling proteins is an important posttranslational mechanism that regulates a number of different biological processes including cell proliferation, differentiation, motility and apoptosis. Although several, well-characterized methods have been developed to quantify the generation of extracellular H2O2, the ability to unequivocally detect and quantify this important signaling molecule within living cells has been relatively limited. Fortunately, a great deal of progress has been made over the past few years in developing H2O2-selective probes that are capable of detecting physiological levels of this signaling molecule. This overview presents a critical evaluation of the established as well as the more recently developed methods to detect and quantify extracellular and intracellular H2O2 produced by living cells.