Protocol for detecting nitrative stress in biological lipid membranes in murine cells and tissues

• Published in: STAR protocols 2024-09, Vol.5 (3), p.103268, Article 103268
• Main Authors: Aggarwal, Tushar, Bellomo, Alyssa, Stevenson, Emily R., Herbert, Julia, Laskin, Debra L., Gow, Andrew J., Izgu, Enver Cagri
• Format: Article
• Language: English
• Subjects: Acute Lung Injury - metabolism; Acute Lung Injury - pathology; Animals; Cell Biology; Chemistry; Lung - metabolism; Lung - pathology; Membrane Lipids - metabolism; Mice; Molecular/Chemical Probes; Peroxynitrous Acid - metabolism; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2024.103268

Detection of nitrative stress is crucial to understanding redox signaling and pathophysiology. Dysregulated nitrative stress, which generates high levels of peroxynitrite, can damage lipid membranes and cause activation of proinflammatory pathways associated with pulmonary complications. Here, we present a protocol for implementing a peroxynitrite-sensing phospholipid to investigate nitrative stress in murine cells and lung tissue. We detail procedures for sensing ONOO– in stimulated cells, both ex vivo and in vivo, using murine models of acute lung injury (ALI). For complete details on the use and execution of this protocol, please refer to Gutierrez and Aggarwal et al.1 [Display omitted] •Probing peroxynitrite in biological lipid environments using DPPC-TC-ONOO–, a synthetic lipid•Nitrative stress was detected by confocal fluorescence microscopy and flow cytometry•Steps for fluorescent staining of organelles in cultured murine cells•Steps for fluorescent detection of peroxynitrite in murine tissue Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Detection of nitrative stress is crucial to understanding redox signaling and pathophysiology. Dysregulated nitrative stress, which generates high levels of peroxynitrite, can damage lipid membranes and cause activation of proinflammatory pathways associated with pulmonary complications. Here, we present a protocol for implementing a peroxynitrite-sensing phospholipid to investigate nitrative stress in murine cells and lung tissue. We detail procedures for sensing ONOO– in stimulated cells, both ex vivo and in vivo, using murine models of acute lung injury (ALI).