Fluorescence-Based Tool To Detect Endogenous Peroxynitrite in M1-Polarized Murine J774.2 Macrophages

Oxidative stress and inflammation are intrinsically linked to each other. In addition, they are implicated in the evolution and progression of noncommunicable diseases (NCDs). Large amounts of reactive oxygen species (ROS) are generated as part of the immune response toward NCDs. Among all of the ROS species, peroxynitrite (ONOO–) has the shortest half-life with <20 ms under typical physiological conditions. Hence, detecting ONOO– and studying its generation in vitro allows for a better understanding of inflammatory processes. We demonstrate that peroxyresorufin-1 (PR1) is a selective and sensitive ONOO– fluorescence-based sensor in J774.2 macrophages. PR1 was able to detect changes in ONOO– production upon investigation of different factors: enhanced generation of ONOO– through LPS and IFN-γ as well as diminished ONOO– production with the introduction of superoxide scavengers and nitric oxide synthase inhibitors. Our study validates PR1 as an effective tool for the detection of ONOO– in J774.2 murine macrophages and should allow for further elucidation of ROS biology and chemistry.