2.11. NADPH Oxidase Activity Measurements

ROS production was measured using the Diogenes-based chemiluminescence kit (National Diagnostics, Atlanta, GA) as before [23,25,26,27]. Shortly, 250,000 PMNs were allowed to adhere to 96-well solid white plates for 1 h at 37 °C in assay medium (previously described). Cells were stimulated by S. aureus isolates (10 MOI), PMA (100 nM) or left unstimulated. Chemiluminescence was measured by a Varioskan Flash microplate luminometer (Thermo Scientific, Waltham, MA, USA) for 90 min. ROS production data are shown as kinetics of representative curves (relative luminescence units, RLU) or integrated superoxide production by analyzing accumulated luminescence for the entire (60 min) or partial (15 min) duration of the measurement and normalizing it on the PMA-stimulated signal as 100%. Superoxide generation of PMNs was specifically tested by the superoxide dismutase-inhibitable cytochrome-c reduction assay as described earlier [24,28]. To measure extracellular superoxide production, PMNs were suspended in assay medium containing 50 μM of cytochrome-c (Sigma, cat#C3131). The cell suspension was added into a 96-well plate and incubated at 37 °C for 5 min in a shaking microplate spectrophotometer Varioskan Flash (ThermoScientific). PMNs were activated with 100 nM PMA, indicated S. aureus CF clinical isolates or Zymosan A particles from Saccharomyces cerevisiae (Sigma, cat#Z4250) opsonized in 10% of autologous serum of the PMN donor at MOI of 10. The increases in absorption at 550 nm were recorded for 60 min with two measurements/min at 37 °C. Superoxide production was calculated with the use of an absorption coefficient of 21 mM⁻¹ cm⁻¹ for cytochrome-c according to the Lambert-Beer law and expressed as nmol O₂^.−/10⁶ PMNs/hr.