Abstract
Cells such as macrophages and neutrophils can internalize a diverse set of particulate matter, illustrated by bacteria and apoptotic bodies through the process of phagocytosis. These particles are sequestered into phagosomes, which then fuse with early and late endosomes, and ultimately with lysosomes to mature into phagolysosomes, through a process known as phagosome maturation. As phagosomes change, they acquire and divest proteins that are associated with the various stages of phagosome maturation. These changes can be assessed at the single-phagosome level by using immunofluorescence methods to study phagosome maturation. Typically, we use indirect immunofluorescence methods that rely on primary antibodies against specific molecular markers that track phagosome maturation. Most commonly, phagosome maturation in macrophages can be determined by staining the cells for Lysosomal-Associated Membrane Protein I (LAMPI) and measuring the fluorescence intensity of LAMPI around each phagosome by microscopy or flow cytometry.
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References
Aderem A (2003) Phagocytosis and the inflammatory response. J Infect Dis 187(0022–1899):S340–S345, Print
García-García E1, and Rosales C. (2002) Signal transduction during Fc receptor-mediated phagocytosis. J Leukoc Biol 72(6):1092–1108
Bongrand P (1994) Relationship between phagosome lysosome fusion, and mechanism. J Leukoc Biol 55:729–734
Flannagan RS, Jaumouillé V, Grinstein S (2012) The cell biology of phagocytosis. Annu Rev Pathol 7:61–98
Vieira OV, Botelho RJ, Rameh L, Brachmann SM, Matsuo T, Davidson HW, Schreiber A, Backer JM, Cantley LC, Grinstein S (2001) Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation. J Cell Biol 155(1):19–25
Kim GHE, Dayam RM, Prashar A, Terebiznik M, Botelho RJ (2014) PIKfyve inhibition interferes with phagosome and endosome maturation in macrophages. Traffic 3:1–21
Huotari J, Helenius A (2011) Endosome maturation. EMBO J 30(17):3481–3500
Vergne I, Fratti RA, Hill PJ, Chua J, Belisle J, Deretic V (2004) Mycobacterium tuberculosis phagosome maturation arrest: mycobacterial phosphatidylinositol analog phosphatidylinositol mannoside stimulates early endosomal fusion. Mol Biol Cell 15:751–760
Aoki V, Sousa JX, Fukumori LMI, Périgo AM, Freitas EL, Oliveira ZNP (2010) Direct and indirect immunofluorescence. An Bras Dermatol 85(4):490–500
St Croix CM, Shand SH, Watkins SC (2005) Confocal microscopy: comparisons, applications, and problems. Biotechniques 39(6):S2–S5
Brown M, Wittwer C (2000) Flow cytometry: principles and clinical applications in hematology. Clin Chem 46(8 Pt 2):1221–1229
Acknowledgements
R.M.D. is supported through a Doctoral Scholarship from the Canadian Institutes of Health Research. R.J.B. is supported through a Canada Research Chair and Early Researcher Awards, and by grants from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada.
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Dayam, R.M., Botelho, R.J. (2017). Quantitative Immunofluorescence to Study Phagosome Maturation. In: Botelho, R. (eds) Phagocytosis and Phagosomes. Methods in Molecular Biology, vol 1519. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6581-6_8
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DOI: https://doi.org/10.1007/978-1-4939-6581-6_8
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