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Infect Immun. 1997 Aug;65(8):3193-8.

Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay.

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1
Department of Medicine, Cornell University Medical College, New York, New York 10021, USA.

Abstract

To compare antibacterial function in macrophages from mice deficient in the respiratory burst oxidase or inducible nitric oxide synthase, we developed a fluorescence-based microplate assay of bacterial survival. As bacteria grow, they convert a formulation of resazurin termed AlamarBlue from its nonfluorescent oxidized state to its fluorescent reduced state. The time required to achieve a given fluorescence is inversely proportional to the number of viable bacteria present when the dye is added. This relationship allows a precise, accurate assessment of bacterial numbers with greater sensitivity and throughput and at less cost than conventional assays. The assay facilitated quantification of the killing of Escherichia coli by S-nitrosoglutathione and hydrogen peroxide and of Salmonella typhimurium by human neutrophils and mouse macrophages. Mouse macrophages lacking the 91-kDa subunit of the respiratory burst oxidase were deficient in their ability to kill S. typhimurium, while those lacking inducible nitric oxide synthase were unimpaired.

PMID:
9234774
PMCID:
PMC175451
[Indexed for MEDLINE]
Free PMC Article
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