Measurement of Phagocytosis and of the Phagosomal Environment in Polymorphonuclear Phagocytes by Flow Cytometry

Elizabeth R. Simons1

1 Boston University School of Medicine, Boston, Massachusetts
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.31
DOI:  10.1002/0471142956.cy0931s51
Online Posting Date:  January, 2010
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Abstract

Phagocytes are the most important early components of the immune response, programmed to recognize, engulf, and destroy immune complexes (formed when antibodies recognize their specific antigens), foreign particles, bacteria, mycobacteria, apoptotic cells, etc. Neutrophils, monocytes, macrophages, and dendritic cells all participate in this process. Flow cytometry permits observation of phagocytes that have responded and, with the appropriate fluorescent probes, of the environment in the phagosome that has enclosed the foreign matter. This unit gives the background and the protocols for performing such studies. Curr. Protoc. Cytom. 51:9.31.1‐9.31.10. © 2010 by John Wiley & Sons, Inc.

Keywords: phagocytosis; phagosome; phagocytes; flow cytometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generic Procedures for OG, DCF, pHrodo, and/or AlexaFluor350 Labeling of Phagocyte Targets
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generic Procedures for OG, DCF, pHrodo, and/or AlexaFluor350 Labeling of Phagocyte Targets

  Materials
  • Dimethyl sulfoxide (DMSO; reagent‐grade) in a closed jar containing Drierite
  • N‐hydroxysuccinimide ester (SE) of desired probes (see recipe) including:
    • DCF SE (Invitrogen/Molecular Probes, cat. no. D2935)
    • pHrodo SE (Invitrogen/Molecular Probes, cat. no. P36600)
    • Alexa Fluor 350 SE (Invitrogen/Molecular Probes, cat. no. A10168)
    • OregonGreen SE (OG SE; Invitrogen/Molecular Probes, cat. no. O6147)
  • Organism, e.g., Mycobacterium avium, or desired organism/stimulus
  • Phosphate‐buffered saline (PBS; see recipe), pH 8 or 9, without glucose, unless otherwise indicated
  • Phosphate‐buffered saline + calcium and magnesium, no glucose (KRP; see recipe), pH 7.4
  • Trypan blue
  • Phagocytes (e.g., purified polymorphonuclear phagocytes) or desired cells, stored in PBS before use, then suspended in KRP, pH 7.4, for 2 min before stimulus is added
  • 1‐, 2‐, 5‐, 15‐, or 50‐ml polycarbonate or polyacrylate conical tubes (rinse tubes with sterile distilled water if tubes were previously sterilized; for storage of probe stocks, use only 1‐ to 2‐ml polycarbonate or glass tubes, not polyethylene or polypropylene)
  • Portable shaker or rocker
  • Refrigerated shaker
  • Benchtop centrifuge
  • 37°C shaking incubator or water bath
  • Flow cytometer with lasers emitting desired excitation wavelengths and appropriate filters and detectors for emission wavelengths
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Figures

Videos

Literature Cited

Literature Cited
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   Brunkhorst, B.A., Lazzari, K.G., Strohmeier, G., Weil, G., and Simons, E.R. 1991. Calcium changes in immune complex‐stimulated human neutrophils. Simultaneous measurement of receptor occupancy and activation reveals full population stimulus binding but subpopulation activation. J. Biol. Chem. 266:13035‐13043.
   Brunkhorst, B.A., Strohmeier, G., Lazzari, K., Weil, G., Melnick, D., Fleit, H.B., and Simons, E.R. 1992. Differential roles of Fc gamma RII and Fc gamma RIII in immune complex stimulation of human neutrophils. J. Biol. Chem. 267:20659‐20666.
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   Miksa, M., Komura, H., Wu, R., Shah, K.G., and Wang, P. 2009. A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester. J. Immunol. Methods 342:71‐77.
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   Ryan, T.C., Weil, G.J., Newburger, P.E., Haugland, R., and Simons, E.R. 1990. Measurement of superoxide release in the phagovacuoles of immune complex‐stimulated human neutrophils. J. Immunol. Methods 130:223‐233.
   Savina, A., Jancic, C., Hughues, S., Guermonprez, P., Vargas, P., Moura, I.C., Lennon‐Dumenil, A‐M., Seabra, M.C., Raposo, G. and Amigorena, S. 2006. NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells. Cell 126:205‐218.
   Seetoo, K.F., Schonhorn, J.E., Gewirtz, A.T., Zhou, M.J., McMenamin, M.E., Delva, L., and Simons, E.R. 1997. A cytosolic calcium transient is not necessary for degranulation or oxidative burst in immune complex‐stimulated neutrophils. J. Leukoc. Biol. 62:329‐340.
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   Strohmeier, G.R., Brunkhorst, B.A., Seetoo, K.F., Bernardo, J., Weil, G.J., and Simons, E.R. 1995a. Neutrophil functional responses depend on immune complex valency. J. Leukoc. Biol. 58:403‐414.
   Strohmeier, G.R., Brunkhorst, B.A., Seetoo, K.F., Meshulam, T., Bernardo, J., and Simons, E.R. 1995b. Role of the Fc gamma R subclasses Fc gamma RII and Fc gamma RIII in the activation of human neutrophils by low and high valency immune complexes. J. Leukoc. Biol. 58:415‐422.
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