Shedding New Light on Detection of Superoxide in Living Cells by Fluorescence Microscopy

摘要

The oxidation of hydroethidine (HE) to ethidium and 2-hydroxyethidium is widely used to detect superoxide in living cells, HE covalently bound to hexyl triphenylphosphonium cation (Mito-HE) accumulates in mitochondria as a function of membrane potential, increasing its selectivity for mitochon-drial sources of superoxide. When live cells were incubated with submicromolar concentrations of probe, the oxidation of Mito-HE occurred in the mitochondria and its oxidation was blocked by the putative superoxide scavenger, FeTCPP. However, loading of endothelial cells with 2-5 muM Mito-HE caused mitochondrial stress, which resulted in relocalization of oxidation products to the cytosol and to the nucleus, Thus, overloading of Mito-HE could lead to artifactual staining. Mito-HE can be used as a sensitive and specific tool for detection of mitochondrial superoxide in living cells when judiciously applied,