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An automated micro-fluorometric assay for monitoring oxidative burst activity of phagocytes

An automated micro-fluorometric assay for monitoring oxidative burst activity of phagocytes

Journal of Immunological Methods 159(1-2): 131-138

A micro-fluorometric assay using 2',7'-dichlorofluorescin diacetate (DCFH-DA) to monitor oxidative burst (OB) in phagocytes has been developed. This assay is based on the oxidation of nonfluorescent DCFH-DA to highly fluorescent 2',7'-dichlorofluorescein (DCF) both intracellularly and extracellularly. A murine macrophage cell line, J774, and a human monocytic cell line, Mono Mac 6, were used as models. The cells were harvested from tissue culture flasks, washed, counted and adjusted to desired concentrations. They were then dispensed into a 96-well flat-bottom tissue culture plate. After adding DCFH-DA and a agent eliciting OB, the plates were incubated in 5% CO-2 at 37 degree C for various periods. The intensity of fluorescence was measured directly in the wells of the tissue culture plate with the cells in situ using a computerized microplate fluorometer at 485 nm excitation and 530 nm emission. This assay provided a rapid measurement of oxidative burst of phagocytes. The automated micro-fluorometric assay may be suitable for screening the immunomodulating activities of various biological and pharmacological substances.

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Accession: 008142184

Download citation: RISBibTeXText

PMID: 8445246

DOI: 10.1016/0022-1759(93)90150-6

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