Mitochondrial ROS Measurement in PBMCs

Similar to our previous studies on LCLs (Rose et al., 2014 (link); Rose et al., 2015a (link); Frye et al., 2016 (link); Frye et al., 2017 (link); Rose et al., 2017 (link); Rose et al., 2018a (link); Bennuri et al., 2019 (link)), PBMCs were exposed to various concentrations of DMNQ (Sigma-Aldrich, St. Louis, MO, United States) for 1 h at 37°C in a non-CO₂ incubator prior to the Seahorse assay. A 5 mg/mL DMNQ solution was diluted in DMEM XF assay media (Agilent Technologies) into a 10X stock and added to cells in an XF-PS plate (Rose et al., 2014 (link)). To verify that mitochondrial reactive oxygen species (ROS) increased as DMNQ concentration increased, mitochondrial superoxide was imaged in PBMCs with increasing DMNQ. MitoSox™ Red (Molecular Probes, Inc., Eugene, OR) was used to depict mitochondrial superoxide production using conditions specified by the manufacturer. MitoSox™ fluorescence was visualized after a 15 min incubation with 0–10 µM DMNQ. Nuclei were counterstained with Hoeschst 33342.