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.
Carey
L A
LA
Department of Physiology, Jefferson Medical College Thomas Jefferson University, Philadelphia, PA 19107, USA.
Schuhl
R A
RA
Gee
M H
MH
D000818:Animals; D000838:Anions; D007700:Kinetics; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D009895:Opsonin Proteins / Q000494:pharmacology*; D012756:Sheep; D013053:Spectrophotometry; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology*
D000838:Anions; D009895:Opsonin Proteins; D013481:Superoxides; D015054:Zymosan; D013755:Tetradecanoylphorbol Acetate