Further characterization of the neutrophil oxidative burst by flow cytometry

The ability to generate reactive oxygen species is essential for neutrophils to kill infectious micro-organisms. We have investigated the 'oxidative burst' in human neutrophils stimulated in vitro with either opsonized zymosan (OZ) or phorbol myristate acetate (PMA) at the single cell level by flow cytometry using dichlorofluorescein diacetate (DCFH-DA) and dihydrorhodamine 123 (DHR) as oxidative probes. These responses have been compared to results obtained with chemiluminescence and spectrophotometric assays. DHR was the most sensitive oxidative probe and PMA the more potent stimulus. The magnitude of the intracellular fluorescence generated with both probes was increased substantially by inhibiting the activities of H2O2-consuming enzymes with a relatively low concentration of azide, which also influenced the distribution of neutrophil subpopulations that expressed fluorescence differentially. Positive correlations between the DCFH-DA and DHR assays were found with both stimuli, and between these assays and ferricytochrome c reduction with OZ-stimulated cells only. We conclude that the DHR flow cytometric assay is the most sensitive technique available for investigating changes that may occur in the oxidative activities and distribution patterns of active neutrophil subpopulations in response to a variety of physiological and pathological conditions.