Nunc f96 microwell polystyrol plate

The sum of intra- and extracellular MPO-derived ROS was measured by using the luminol-amplified chemiluminescence assay. Neutrophils (400,000 cells/sample) were resuspended in complete medium without FCS and seeded in a flat-bottom white 96-well plate (NuncTM F96 MicroWellTM polystyrol plate, Thermo Fisher). Subsequently, 60 μM luminol (Invitrogen, Germany) was added, and the cells were stimulated by the addition of 20 nM phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich). The chemiluminescence resulting from ROS release was analyzed immediately by an Infinite M200pro-Tecan reader (Tecan, Männedorf, Switzerland) and Tecan i-control 1.7 software. ROS release was monitored every minute for a period of 1 h at 37°C and 5% CO₂.

The sum of intra- and extracellular MPO-derived ROS (28 ) was measured by using a luminol-amplified chemiluminescence assay. Polarized neutrophils (400,000 cells/sample) were resuspended in complete medium without FCS and seeded in a flat-bottom white 96-well plate (Nunc^TM F96 MicroWell^TM polystyrol plate, Thermo Fisher). Subsequently, 60 μM luminol (Invitrogen, Germany) was added, and the cells were stimulated by the addition of 20 nM phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich). The chemiluminescence resulting from ROS release was analyzed immediately by an Infinite M200pro-Tecan reader (Tecan, Männedorf, Switzerland) and Tecan i-control 1.7 software. ROS release was monitored every minute for a period of 1 h at 37°C and 5% CO₂. Extracellular superoxide was detected by using a lucigenin-amplified chemiluminescence assay (28 ). This assay was performed the same way as the luminol assay, but with 0.2 mM lucigenin (Alexis, Loerrach, Germany) instead of luminol.