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.