EVALUATION OF BACTERIAL SURVIVAL AND PHAGOCYTE FUNCTION WITH A FLUORESCENCE-BASED MICROPLATE ASSAY

To compare antibacterial function in macrophages from mice deficient i n the respiratory burst oxidase or inducible nitric oxide synthase, we developed a fluorescence-based microplate assay of bacterial survival . iis bacteria grow, they convert a formulation of resazurin termed Al amarBlue from its nonfluorescent oxidized state to its fluorescent red uced state, The time required to achieve a given fluorescence is inver sely proportional to the number of viable bacteria present when the dy e 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 o f 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 resp iratory burst oxidase were deficient in their ability to kill S. typhi murium, while those lacking inducible nitric oxide synthase were unimp aired.