2.5. Measurement of Reactive Oxygen Species (ROS)

Freshly isolated neutrophils were dispensed in triplicate into a 96-well microplate, pretreated with or without 100 ng/mL recombinant human IL-18 (rhIL-18) (InvivoGen, San Diego, CA, USA) for 2 h at 37 °C. Then, 1 μM dihydrorhodamine 123 (DHR123) (Invitrogen, Eugene, OR, USA) was added to the wells for 15 min before stimulation with 100 nM N-formyl-methionyl-leucyl-phenylalanine (fMLP) (Sigma-Aldrich, St. Louis, MO, USA). For the blocking assay, 10 μg/mL polyclonal anti-IL18RAP antibody (Invitrogen, Rockford, IL, USA) or goat IgG isotype control (Novus Biologicals, Centennial, CO, USA) was incubated with neutrophils for 20 min at 37 °C prior to stimulation with rhIL-18, DHR123, and fMLP, as described above. Fluorescence signal (FS) was detected in real time at 529 nm and recorded at 2- to 5-min intervals for 30 min using a CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany). Enhancement in fMLP-mediated ROS generation at 30 min was calculated using the formula: (FS[rhIL18+fMLP]−FS[fMLP])/FS[fMLP] × 100%.