A Rapid Microplate-Based Fluorometric Assay for Phagocytosis

Abstract

A highly sensitive quantitative fluorometric assay for phagocytosis, previously measured using fluorescence spectrophotometry or flow cytometry, has been adapted for use with a 96-well fluorescence plate reader. The technique allows rapid analysis of large numbers of samples, and requires only a small sample volume. Comparison of plate types demonstrated that opaque white 96-well luminostrips produced a 100 fold greater fluorescent output, and were more sensitive than black fluoroplates. Intraplate variability was also significantly lower using white luminostrips. For the phagocytic assay, fluorescein conjugated polystyrene beads were incubated with macrophage monolayers in white luminostrips. After incubation, cells were washed, lysed and phagocytosis quantified by determining the fluorescent intensity using a fluorescence plate reader. The number of beads phagocytized was determined from a standard curve of bead number versus fluorescent output. The phagocytic activity of resident and thioglycollate-elicited peritoneal macrophages was compared using this technique.