Measurement of Mitochondrial Superoxide

mROS was measured using the MitoSOX™ Red mitochondrial superoxide indicator (M36008, Thermo Fisher, USA). MitoSOX is a novel fluorogenic probe for sensitive selective detection of superoxide (instead of reactive nitrogen species) in the mitochondria of live cells. Once in the mitochondria, the MitoSOX reagent is oxidized by superoxide and exhibits red fluorescence. The left lung tissues after modeling from different groups were immediately cut and digested with collagenase type I (17018029, Thermo Fisher, USA) on a shaker at 37°C for 30 min. The samples were filtered two times using a cell mesh, and then the red blood cells were lysed with red blood cell lysis buffer (R1010, Solarbio, China), and followed by centrifugation to obtain the single cells. The obtained mouse single cells or RAW264.7 cells after modeling in vitro were seeded into 24-well plates at a density of 5 × 10⁵ cells/mL. After cells had adhered to the wells, the cells were incubated with MitoSOX (5 μm) for 10 min in the dark and nuclei were visualized by staining with Hoechst 3342 (C0030, Solarbio, China) for 20 min at room temperature as previously described [35 (link)]. The stained cells were then washed with DMEM and observed using a fluorescence microscope (EVOS FL AutoLife Technologies).