Quantifying Inflammatory Cell ROS Production

ROS production was measured using L-012-enhanced chemiluminescence, as previously described (To et al., 2017 (link)). Inflammatory cells isolated from the BAL were seeded into a 96-well OptiView plate (5 × 10⁴ cells/well) with Dulbecco’s Modified Eagle’s Medium (DMEM; Thermofisher, United States) containing 4.5 g/L of glucose, 110 mg of sodium pyruvate and 10% Fetal Bovine Serum (FBS; Sigma-Aldrich, United States), and allowed to adhere for 3 h prior to starting the assay. Cells were then washed with warm 37°C Krebs-HEPES buffer and exposed to a Krebs-HEPES buffer containing L-012 (10⁻⁴ mol/L) (WAKO Chemicals) in the absence (i.e., basal ROS production) or presence (stimulated ROS production) of the protein kinase C (PKC) and NADPH oxidase activator, phorbol 12,13-dibutyrate (PDB; 10⁻⁶ mol/L) (Sigma-Aldrich, United States). The same treatments were performed in blank wells (i.e. with no cells), which served as controls for background luminescence. All treatment groups were performed in triplicate. Photon emission [relative light units (RLU)/s] was detected using the BMGlabtech microplate reader (CLARIOstar, Germany) and recorded from each well for 1 s over 60 cycles. Individual data points for each group were derived from the average values of the three replicates minus the respective blank controls.