Amplex red reagent

ROS production was determined using a10-acetyl-3,7-dihydroxyphenoxazine-based plate assay (Amplex™ Red Reagent, Invitrogen, Carlsbad, CA, USA). The assay was conducted in opaque, 96-well plates (Microlite™ 1+ White Microtiter™ Plate, Sigma-Aldrich, St. Louis, MO, USA). For each donor, six stimulatory conditions were tested in triplicates. For each condition, 50 μL of the 4 × 10 6 cells/mL suspension, 50 μL of the antibiotic of interest in the indicated concentrations or PBS as control, 50 μL of the Amplex™ Red Reagent (final concentration of 0.625 μM), 20 μL horseradish peroxidase (final concentration 1/160 U/mL; Sigma-Aldrich), and 50 μL of the indicated stimulus or PBS were carefully combined. All unstimulated conditions consisted of 100 µL PBS, 50 µL of PMNs (4 × 10 6 cells/mL), and 50 µL AR (0.625 µM), only differing by the presence or absence of 20 µL horseradish peroxidase in order to determine basal burst activity. To continuously detect the converted fluorescent substrate resorufin within the Amplex™ Red assay, the LB 940 Multimode Reader Mithras (Berthold Technologies, Bad Wildbad, Germany) was used to measure for a total of 60 min therefore accounting for 52 measurements per well in 96-well plates. An excitation filter of 570 nm and an emission filter of 615 nm wavelength were applied. Measurements were taken at 37°C.