Biomaterial Database

Further characterization of the neutrophil oxidative burst by flow cytometry. 1993

J A Smith, and M J Weidemann

Division of Biochemistry and Molecular Biology, School of Life Sciences, Faculty of Science, Australian National University, Canberra.

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.

UI	MeSH Term	Description	Entries
D008297	Male		Males
D009504	Neutrophils	Granular leukocytes having a nucleus with three to five lobes connected by slender threads of chromatin, and cytoplasm containing fine inconspicuous granules and stainable by neutral dyes.	LE Cells,Leukocytes, Polymorphonuclear,Polymorphonuclear Leukocytes,Polymorphonuclear Neutrophils,Neutrophil Band Cells,Band Cell, Neutrophil,Cell, LE,LE Cell,Leukocyte, Polymorphonuclear,Neutrophil,Neutrophil Band Cell,Neutrophil, Polymorphonuclear,Polymorphonuclear Leukocyte,Polymorphonuclear Neutrophil
D005434	Flow Cytometry	Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake.	Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D005452	Fluoresceins	A family of spiro(isobenzofuran-1(3H),9'-(9H)xanthen)-3-one derivatives. These are used as dyes, as indicators for various metals, and as fluorescent labels in immunoassays.	Tetraiodofluorescein
D005456	Fluorescent Dyes	Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags.	Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012235	Rhodamines	A family of 3,6-di(substituted-amino)-9-benzoate derivatives of xanthene that are used as dyes and as indicators for various metals; also used as fluorescent tracers in histochemistry.	Rhodamine
D013053	Spectrophotometry	The art or process of comparing photometrically the relative intensities of the light in different parts of the spectrum.
D016897	Respiratory Burst	A large increase in oxygen uptake by neutrophils and most types of tissue macrophages through activation of an NADPH-cytochrome b-dependent oxidase that reduces oxygen to a superoxide. Individuals with an inherited defect in which the oxidase that reduces oxygen to superoxide is decreased or absent (GRANULOMATOUS DISEASE, CHRONIC) often die as a result of recurrent bacterial infections.	Oxidative Burst,Burst, Oxidative,Burst, Respiratory,Bursts, Oxidative,Bursts, Respiratory,Oxidative Bursts,Respiratory Bursts
D017382	Reactive Oxygen Species	Molecules or ions formed by the incomplete one-electron reduction of oxygen. These reactive oxygen intermediates include SINGLET OXYGEN; SUPEROXIDES; PEROXIDES; HYDROXYL RADICAL; and HYPOCHLOROUS ACID. They contribute to the microbicidal activity of PHAGOCYTES, regulation of SIGNAL TRANSDUCTION and GENE EXPRESSION, and the oxidative damage to NUCLEIC ACIDS; PROTEINS; and LIPIDS.	Active Oxygen Species,Oxygen Radical,Oxygen Radicals,Pro-Oxidant,Reactive Oxygen Intermediates,Active Oxygen,Oxygen Species, Reactive,Pro-Oxidants,Oxygen, Active,Pro Oxidant,Pro Oxidants,Radical, Oxygen