Analysis of Macrophage Phagocytosis: Quantitative Assays of Phagosome Formation and Maturation Using High-Throughput Fluorescence Microscopy
Phagocytosis of invading pathogens by macrophages represents a fundamental component of the innate immune system. In this chapter, we describe protocols designed for high-throughput analysis of phagosome formation and maturation using latex beads as model phagocytic targets. The method takes advantage of an automated fluorescence microscope platform to investigate Fcγ receptor-mediated particle internalization. First, procedures to opsonize and fluorescently label the model particles are outlined. In combination with the robotic fluorescence microscope, these labeling methods provide for the quantitative high-throughput assessment of phagocytosis. Acidification of the phagosomal lumen can be used as an index of maturation. We describe a fluorimetric procedure to assess phagosomal pH based on the partition of a membrane-permeant weak base that accumulates in acidic intracellular compartments. Lastly, a description of the hardware and software components of the robotic high-throughput fluorescence microscope platform is provided.
KeywordsPhagocytosis Acidification Vacuolar pH Fcγ receptor Macrophage
The authors thank Christopher Fladd for his assistance with the Cellomics KineticScan HSC Reader, as well as the SIDNET facility at the Hospital for Sick Children. B.E.S. is supported by a studentship from the McLaughlin Center for Molecular Medicine. S.G. is the current holder of the Pitblado Chair in Cell Biology.
Research in the authors' laboratory is supported by the Heart and Stroke Foundation of Canada, the Canadian Cystic Fibrosis Foundation, and the Canadian Institutes of Health Research.