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 (O-2(-)) production from opsonized zymosan-stimulated sheep poly morphonuclear leukocytes (PMNs). This method for use with the particul ate neutrophil activator is a modification of the cytochrome c reducti on system that measures O-2(-) release from adherent neutrophils stimu lated,with soluble mediators such as phorbol-myristate-acetate (PMA). We measured O-2(-) release from PMNs (250000 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 produc tion while having little effect on the optical density of the reagent blank. O-2(-) values Obtained using this method were nor statistically different from values measured using cell-fee supernatants. This syst em was useful in demonstrating PMN priming, as evidenced by the 2.7 ti mes increase in O-2(-) production above baseline from neutrophils isol ated from sheep given a 12-hour low-dose endotoxin infusion. This is a convenient and sensitive method for measurement of neutrophil O-2(-) production in response to a receptor-mediated, particulate stimulus.