Advertisement

Analysis of Macrophage Phagocytosis: Quantitative Assays of Phagosome Formation and Maturation Using High-Throughput Fluorescence Microscopy

  • Benjamin E. Steinberg
  • Sergio GrinsteinEmail author
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 531)

Summary

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.

Keywords

Phagocytosis Acidification Vacuolar pH Fcγ receptor Macrophage 

Notes

Acknowledgments

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.

References

  1. 1.
    Vieira, O. V., Botelho, R. J., and Grinstein, S. (2002) Phagosome maturation: aging gracefully. Biochem J 366, 689–704.PubMedGoogle Scholar
  2. 2.
    Lukacs, G. L., Rotstein, O. D., and Grinstein, S. (1990) Phagosomal acidification is mediated by a vacuolar-type H(±)-ATPase in murine macrophages. J Biol Chem 265, 21099–21107.PubMedGoogle Scholar
  3. 3.
    Lukacs, G. L., Rotstein, O. D., and Grinstein, S. (1991) Determinants of the phagosomal pH in macrophages. In situ assessment of vacuolar H(+)-ATPase activity, counterion conductance, and H+ “leak”. J Biol Chem 266, 24540–24548.PubMedGoogle Scholar
  4. 4.
    Gordon, A. H., Hart, P. D., and Young, M. R. (1980) Ammonia inhibits phagosome–lysosome fusion in macrophages. Nature 286, 79–80.PubMedCrossRefGoogle Scholar
  5. 5.
    Pepperkok, R., and Ellenberg, J. (2006) High-throughput fluorescence microscopy for systems biology. Nat Rev Mol Cell Biol 7, 690–696.PubMedCrossRefGoogle Scholar
  6. 6.
    Steinberg, B. E., Scott, C. C., and Grinstein, S. (2006) High-throughput assays of phagocytosis, phagosome maturation, and bacterial invasion. Am J Physiol Cell Physiol 292, C945–C952.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Cell Biology Program, Hospital for Sick ChildrenTorontoCanada

Personalised recommendations