Luminol

A chemiluminescence-based assay was used to determine ROS production by neutrophils. Cells were suspended in RPMI-1640 medium without phenol red at a final concentration of 1.5 x 10⁶ cells/ml and were pre-incubated at 37°C in the presence or absence of the priming agent TNF-α (50 ng/ml). Following 10 minutes of incubation, cells were added to a white, clear-bottom, 96-well microtiter plate (PerkinElmer) and stimulated with PMA (150 ng/ml), ultrapure LPS from E. coli (10 µg/ml), PGN from S. aureus (10 µg/ml), CpG oligodeoxynucleotides (3 µM), TNF-α (50 ng/ml) or IL-1β (500 ng/ml) in the presence of 5 mM luminol (Sigma-Aldrich). Kinetic measurements of luminol oxidation were performed during 1.5 hours at 37°C using a Clariostar Monochromator Microplate Reader (BMG Labtech, Orthenberg, Germany). The results were subtracted by values obtained with PMA stimulation in the absence of luminol.

Mouse spleens were gently meshed using a 40 μM cell strainer and erythrocytes were lysed with ACK lysing buffer (GIBCO). Neutrophils were isolated via negative selection with the EasySep mouse neutrophil isolation kit (STEMCELL Technologies). A total of 1 × 10⁵ neutrophils were then incubated with the chemiluminescent probe luminol (Sigma) and horseradish peroxidase (HRP; Sigma) and stimulated with 100 nM PMA (Sigma). Reactive oxygen species (ROS) production was measured by HRP mediated oxidation of luminol, producing a chemiluminescent signal that is detected with an UV filter on a spectrophotometric microplate reader (Fluostar Omega, BMG labtech).