Molecular visualizing and quantifying immune-associated peroxynitrite fluxes in phagocytes and mouse inflammation...

Reactions of peroxynitrite (ONOO^-) with biomolecules can lead to cytotoxic and cytoprotective events. Due to the difficulty of directly and unambiguously measuring its levels, most of the beneficial effects associated with ONOO^- in vivo remain controversial or poorly characterized. Recently, optical imaging has served as a powerful noninvasive approach to studying ONOO^- in living systems. However, ratiometric probes for ONOO^- are currently lacking. Herein, we report the design, synthesis, and biological evaluation of F₄₈₂, a novel fluorescence indicator that relies on ONOO^--induced diene oxidation. The remarkable sensitivity, selectivity, and photostability of F₄₈₂ enabled us to visualize basal ONOO^- in immune-stimulated phagocyte cells and quantify its generation in phagosomes by high-throughput flow cytometry analysis. With the aid of in vivo ONOO^- imaging in a mouse inflammation model assisted by F₄₈₂, we envision that F₄₈₂ will find widespread applications in the study of the ONOO^- biology associated with physiological and pathological processes in vitro and in vivo.