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. 1997 Aug;65(8):3193–3198. doi: 10.1128/iai.65.8.3193-3198.1997

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

M U Shiloh 1, J Ruan 1, C Nathan 1
PMCID: PMC175451  PMID: 9234774

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.

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Selected References

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