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

•F482 was applied in ratiometrically detecting ONOO-.•Carboxylic BODIPY formation by ONOO--induced diene oxidation was confirmed.•F482 enables us to quantify ONOO- in phagosomes by high-throughput flow cytometry.•Visualizing and quantifying ONOO- fluxes in mouse inflammation model was realized.

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.