Detection of Mitochondrial ROS Formation Using Live Cell Imaging and Flow Cytometry

Sixty thousand freshly isolated bone marrow derived murine neutrophils were seeded into a 96-well glass bottom plate per well in 1 × HBSS, supplemented with 0.01% BSA, w/o CaCl₂, w/o MgCl₂ and allowed to settle for 45 min at 37°C, following priming with 20 ng/ml TNFα (Peprotech, Rocky Hill, NJ, USA) for 20 min at 37°C. Afterwards, supernatants were removed and 5 μM of the mitochondrial ROS indicator MitoSOX (Thermo Fisher Scientific) was added followed by incubation for 30 min at 37°C. Supernatants were removed and cells were left untreated, or stimulated with 100 nM PMA, 5 μM ionomycin or 10 μM casin in 1 × HBSS supplemented with 0.01% BSA, 2 mM CaCl₂, 2 mM MgCl₂. The generation of mitochondrial ROS over time was detected using the Incucyte® Live-Cell Analysis System (Essen BioScience) for 1 h at 37°C every 4 min. The amount of mitochondrial ROS produced over time was analyzed using the IncuCyte Zoom elements 2000 software. To detect mitochondrial ROS formation in primary human neutrophils, 5 × 10⁵ freshly isolated neutrophils per sample were pre-incubated with 5 μM MitoSOX for 30 min. at 37°C. Cells were left untreated or incubated with PMA (20 nM), ionomycin (10 μM) or the Cdc42 inhibitor casin (10 μM) for 1 h at 37°C. The fluorescence signal of MitoSOX was analyzed using flow cytometry.