Microplate reader assay for measurement of opsonized zymosan-stimulated superoxide anion production by neutrophils

We report a microplate reader assay for the measurement of superoxide anion (O2-) production from opsonized zymosan-stimulated sheep polymorphonuclear leukocytes (PMNs). This method, for use with the particulate neutrophil activator, is a modification of the cytochrome c reduction system that measures O2- release from adherent neutrophils stimulated with soluble mediators such as phorbol-myristate-acetate (PMA). We measured O2- release from PMNs (250,000 per well) stimulated with opsonized zymosan over a dose range from 0.0625 mg/mL to 0.5 mg/mL. A dose of 0.125 mg/mL was shown to stimulate maximal superoxide production while having little effect on the optical density of the reagent blank. O2- values obtained using this method were not statistically different from values measured using cell-free supernatants. This system was useful in demonstrating PMN priming, as evidenced by the 2.7 times increase in O2- production above baseline from neutrophils isolated from sheep given a 12-hour, low-dose endotoxin infusion. This is a convenient and sensitive method for measurement of neutrophil O2- production in response to a receptor-mediated, particulate stimulus.