Centro microplate luminometer lb960

PMN and PBMC (1 × 10⁶ cells/mL) were preincubated at 37°C in supplemented PBS with the test compound for 10 min. Next, lucigenin (10 μM) and PMA (100 nM) or opsonised zymosan (1 mg/mL) were added and the light emission was measured for 30 min at 37°C using a plate luminometer (Centro Microplate Luminometer LB960, Berthold Technologies, Oak Ridge, TN, USA). The final assay volume was 250 μL. The integrated light emission was used as an analytical parameter to measure the superoxide anion produced by the stimulated cells. The inhibitory potency was calculated using the light emission generated by the control, in which activated cells were incubated in the absence of the test compounds as [26 (link)].

PMN cells (1 × 10⁶ cells per mL) in the absence (control) and presence of the studied compounds were pre-incubated in HBSS at 37 °C for 10 min using a flat bottom white microplate. Then, luminol (10 μmol L⁻¹) was added, and the reaction was triggered by adding PMA (0.1 μmol L⁻¹). The light emission was measured for 30 min at 37 °C using a plate luminometer (Centro Microplate Luminometer LB960, Berthold Technologies, Oak Ridge, TN, USA). The final reaction volume was 250 μL. The total ROS production was evaluated by luminol-enhanced chemiluminescence.^{43 (link)} The integrated light emission was used as an analytical parameter for the measurement of total ROS production. The inhibitory potency was calculated using the light emission generated by the positive control (100%) as reference.

Consumption of NCT and HOCl were monitored by their absorbances at 252 and 290 nm, respectively, using a Perkin Elmer Lambda 25 UV–visible spectrophotometer (Shelton, CT, USA). The reaction mixtures were composed of 0.25 mM NCT or HOCl, and 0.5 mM H₂O₂ in 0.1 M phosphate buffer, pH 7.1 and 37 °C. The production of ¹O₂ was monitored by its dimol light emission or, indirectly, by the light emission generated by its reaction with melatonin using a plate luminometer (Centro Microplate Luminometer LB960, Berthold Technologies, Oak Ridge, TN, USA). The reaction mixtures were composed of 1.0 mM NCT or HOCl, 20 mM H₂O₂, in the presence or absence of 1 mM melatonin in 0.1 M phosphate buffer, pH 7.1 and 37 °C. The reactions were triggered by the addition of H₂O₂.