Superoxide anion assay kit

Superoxide anion levels were assessed using a Superoxide Anion Assay Kit (Sigma-Aldrich Chemical Co), according to manufacturer's instruction relative to testing changes in superoxide anion production directly on intact cells.

Superoxide anion (O₂^•−) production was detected using the luminol-based Superoxide Anion Assay Kit (catalog no. CS1000, Sigma-Aldrich, St. Louis, MO). Six cell lines with appreciable NOX1 mRNA expression (LS513, NCI-H508, LS174T, HT-29, WiDr, and HEK293-NOX1) and three cell lines with undetectable NOX1 expression (HCT-116, RKO, and SW620) were harvested in log-phase growth and counted. Following the manufacturer's instructions, cells were resuspended in assay medium at a density of 2x10⁶ cells/ml for HEK293-vector and HEK293-NOX1, and 10⁷ cells/ml for all other cell lines. One hundred microliter of each cell suspension was added per well in a 96-well plate. Before starting measurements, 100 μl of assay buffer containing 3 μl of luminol solution, 3 μl of enhancer solution was added to each well, in the presence or absence of 200 nM phorbol 12-myristate 13-acetate (PMA, catalog no. P8139, Sigma-Aldrich, St. Louis, MO) and 200 U/ml superoxide dismutase-polyethylene glycol from bovine erythrocytes (PEG-SOD, catalog no. S9549, Sigma-Aldrich, St. Louis, MO). The samples were mixed and placed immediately into a GloMax^® Microplate Luminometer (Promega BioSciences, San Luis Obispo, CA), and the luminescence was measured at 37°C during a 1.5–2 h period of observation. Every experiment was performed in triplicate.

Isolated neutrophils were placed in black culture 96-well plates in RPMI-1640 culture medium, and pre-incubated with LAS194046 (0.01 nM–10 µM), fluticasone propionate (0.01 nM–10 µM) and their combinations for 30 min, and then stimulated with fMLP 1 µM between 0 to 45 min. The fMLP stimulus was selected for superoxide anion formation because its robust induction on the reactive oxygen species that is reproducible in different models of pharmacology testing^{16 ,20 (link),21 (link)}.
Superoxide anions were determined based on chemiluminescence from luminol with the Superoxide Anion Assay Kit (Sigma Aldrich cat. no. CS1000) following the instructions of the manufacturer. In brief, neutrophils were incubated with different drugs, assay buffer, luminol solution and enhancer solution as specified by manufacturer, during 30 min, followed by the stimulation with fMLP, In presence of an “enhancer” superoxide anions convert luminol into a reactive peroxide that in several transitions emits energy as a photon producing a blue glow (chemiluminescence) that has been tested during 4 h as indicate manual instructions. Chemiluminescence was measured each 5 min, and Area Under Curve (AUC) were calculated for all time-dependent curves of superoxide anion during 45 min of fMLP stimulation.