Abstract
Neutrophils are short-lived granulocytes essential for innate host defense. We describe here methods for analysis of resting and activated cells using immunofluorescence and confocal microscopy. Procedures for stimulation of adherent and suspended cells are provided along with protocols for particle opsonization and synchronized phagocytosis. Most importantly, we describe in detail methods for rapid and efficient cell fixation and permeabilization that optimize detection of granule proteins and NADPH oxidase components. Variables that impact antigen detection (such as cell spreading, degranulation, and phagocytosis) are discussed as are methods for image acquisition and analysis.
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References
Faurschou, M. and Borregaard, N. (2003) Neutrophil granules and secretory vesicles in inflammation. Microbes Infect. 5, 1317–1327.
Nauseef, W. M. (2004) Assembly of the phagocyte NADPH oxidase. Histochem. Cell Biol. 122, 277–291.
Allen, L.-A. H. and Aderem, A. (1995) A role for MARCKS, the α isozyme of protein kinase C and myosin I in zymosan phagocytosis by macrophages. J. Exp. Med. 182, 829–840.
Allen, L.-A. H. and Aderem, A. (1996) Molecular definition of distinct cytoskeletal structures involved in complement-and Fc receptor-mediated phagocytosis in macrophages. J. Exp. Med. 184, 627–637.
Allen, L.-A. H., Schlesinger, L. S., and Kang, B. (2000) Virulent strains of Helicobacter pylori demonstrate delayed phagocytosis and stimulate homotypic phagosome fusion in macrophages. J. Exp. Med. 191, 115–127.
Allen, L.-A. H., DeLeo, F. R., Gallois, A., Toyoshima, S., Suzuki, K., and Nauseef, W. M. (1999) Transient association of the nicotinamide adenine dinucleotide phosphate oxidase subunits p47phox and p67phox with phagosomes in neutrophils from patients with X-linked chronic granulomatous disease. Blood 93, 3521–3530.
Allen, L.-A. H., Beecher, B. R., Lynch, J. T., Rohner, O. V., and Wittine, L. M. (2005) Helicobacter pylori disrupts NADPH oxidase targeting in human neutrophils to induce extracellular superoxide release. J. Immunol. 174, 3658–3667.
DeLeo, F. R., Allen, L.-A. H., Apicella, M., and Nauseef, W. M. (1999) NADPH oxidase activation and assembly during phagocytosis. J. Immunol. 163, 6732–6740.
Boyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. J. Clin. Lab. Invest. 21, 77–89.
Burritt, J. B., Quinn, M. T., Jutila, M. A., Bond, C. W., and Jesaitis, A. J. (1995) Topological mapping of neutrophil cytochrome b epitopes with phage-display libraries. J. Biol. Chem. 270, 16,974–16,980.
Burritt, J. B., Busse, S. C., Gizachew, D., et al. (1998) Antibody imprint of a membrane protein surface. Phagocyte flavocytochrome b. J. Biol. Chem. 273, 24,847–24,852.
Nauseef, W. M., Root, R. K., and Malech, H. L. (1983) Biochemical and immunological anaysis of hereditary myeloperoxidase deficiency. J. Clin. Invest. 71, 1297–1303.
Schwartz, J. T. and Allen, L.-A. H. (2006) Role of urease in megasome formation and Helicobacter pylori survival in macrophages. J. Leukoc. Biol. 79, 1214–1225.
Botelho, R. J., Tapper, H., Furuya, W., Mojdami, D., and Grinstein, S. (2002) FcβR-mediated phagocytosis stimulates localized pinocytosis in human neutrophils. J. Immmunol. 169, 4423–4429.
Melan, M. A. and Sluder, G. (1992) Redistribution and differential extraction of soluble proteins in permeabilized cultured cells: implications for immunofluorescence microscopy. J. Cell Sci. 101, 731–743.
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© 2007 Humana Press Inc., Totowa, NJ
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Allen, LA.H. (2007). Immunofluorescence and Confocal Microscopy of Neutrophils. In: Quinn, M.T., DeLeo, F.R., Bokoch, G.M. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology™, vol 412. Humana Press. https://doi.org/10.1007/978-1-59745-467-4_18
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DOI: https://doi.org/10.1007/978-1-59745-467-4_18
Publisher Name: Humana Press
Print ISBN: 978-1-58829-788-4
Online ISBN: 978-1-59745-467-4
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