Spatial, temporal, and quantitative manipulation of intracellular hydrogen peroxide in cultured cells

Hydrogen peroxide (H₂O₂) is produced endogenously in a number of cellular compartments, including the mitochondria, the endoplasmic reticulum, peroxisomes, and at the plasma membrane, and can play divergent roles as a second messenger or a pathological toxin. It is assumed that the tuned production of H₂O₂ within neuronal and nonneuronal cells regulates a discreet balance between survival and death. However, a major challenge in understanding the physiological versus pathological role of H₂O₂ in cells has been the lack of validated methods that can spatially, temporally, and quantitatively modulate H₂O₂ production. A promising means of regulating endogenous H₂O₂ is through the expression of peroxide-producing enzyme d-amino acid oxidase (DAAO from Rhodotorula gracilis lacking a peroxisomal targeting sequence). Using viral vectors to express DAAO in distinct cell types and using targeting sequences to target DAAO to distinct subcellular sites, we can manipulate H₂O₂ production by applying the substrate d-alanine or permeable analogs of d-alanine. In this chapter, we describe the use of DAAO to produce H₂O₂ in culture models and the real-time visual validation of this technique using two-photon microscopy and chemoselective fluorescent probes.