Rapid microassays of phagocytosis, bacterial killing, superoxide and hydrogen peroxide production by human neutrophils in vitro

Abstract

Simple, rapid microassays for simultaneous measurement of phagocytosis, bacterial killing, superoxide and hydrogen peroxide production by human neutrophils in vitro are described. All assays employ 96-well flat bottom tissue culture plates which were incubated on a microtitre plate shaker at 37 degrees C. The separate evaluation of ingestion and intracellular killing of E. coli and S. aureus was based on the incorporation of [3H]uridine into viable extracellular bacteria. There was good correlation between plate counts of viable bacteria and amount of radiolabel incorporation. Phagocytosis and killing can be measured in a maximum of 100 microliter reaction mixture, requiring only 2.5 X 10(5) neutrophils per test and the assay is complete within 60 min. Assay of superoxide production by stimulated neutrophils was based on superoxide-dependent reduction of ferricytochrome c as measured spectrophotometrically at 550 nm in wells of tissue culture plates containing 150 microliter of reaction mixture. The assay requires only 1.25 X 10(5) neutrophils per test and is complete within 50 min. Quantitation of hydrogen peroxide was based on horseradish peroxidase-dependent oxidation of phenol red. The technique is as for superoxide detection except that the reaction must be terminated by the addition of 1 M NaOH at the desired time intervals. None of the assays require sampling during the incubation period. The principal advantages of the described techniques are increased simplicity and speed, requirement of low numbers of neutrophils and applicability to analysis of large number of samples in parallel.