title: Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay.
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.
authors.affiliation: Department of Medicine, Cornell University Medical College, New York, New York 10021, USA.
authors.forename: M U
authors.initials: MU
authors.lastname: Shiloh
authors.forename: J
authors.initials: J
authors.lastname: Ruan
authors.forename: C
authors.initials: C
authors.lastname: Nathan
mesh_terms: D000818:Animals; D001770:Blood Bactericidal Activity; D005453:Fluorescence; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000502:physiology; D009603:Nitroso Compounds / Q000494:pharmacology; D026422:S-Nitrosoglutathione; D012486:Salmonella typhimurium / Q000276:immunology*
chemical_list: D009603:Nitroso Compounds; D026422:S-Nitrosoglutathione; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D005978:Glutathione