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 Fcgamma 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.
Cell Biology Program, Hospital for Sick Children, Toronto, ON, Canada, M5G 1X8.
Benjamin E
BE
Steinberg
Sergio
S
Grinstein
D000818:Animals; D001331:Automation; D001681:Biological Assay / Q000379:methods*; D002454:Cell Differentiation; D006801:Humans; D008264:Macrophages / Q000166:cytology*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D008863:Microspheres; D009895:Opsonin Proteins / Q000378:metabolism; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism*; D013194:Staining and Labeling
D009895:Opsonin Proteins