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

  • 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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Literature Cited

Literature Cited
   Bellaire, B.H., Roop, R.M. II, and Cardelli, J.A. 2005. Opsonized virulent Brucella abortus replicates within nonacidic, endoplasmic reticulum‐negative, LAMP‐1‐positive phagosomes in human monocytes. Infect. Immun. 73:3702‐3713.
   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.
   Elbim, C. and Lizard, G. 2009. Flow cytometric investigation of neutrophil oxidative burst and apoptosis physiological and pathological situation. Cytometry Part A 75:475‐481.
   Fuhrmann, S., Streitz, M., and Kern, F. 2008. How flow cytometry is changing the study of TB immunology and clinical diagnosis. Cytometry Part A 73:1100‐1106.
   Guillemot, J‐C., Kruskal, B.A., Adra, C.N., Zhu, S., Ko, J‐L., Burch, P., Nocka, K., Seetoo, K., Simons, E., and Lim, B. 1996. Targeted disruption of GDP‐dissociation inhibitor for Rho‐related proteins, GDID4: Normal hematopoietic differentiation by subtle defect in superoxide production by macrophages derived from in vitro embryonal stem cell differentiation. Blood 88:2722‐2731.
   Herrmann, J.M., Bernardo, J., Long, H.J., Seetoo, K., McMenamin, M.E., Batista, E.L. Jr., Van Dyke, T.E. and Simons, E.R. 2007. Sequential chemotactic and phagocytic activation of human polymorphonuclear neutrophils. Infect. Immun. 75:398‐3998.
   Heinzelmann, M., Herzig, D.O., Swain, B., Mercer‐Jopnes, M.A., Bergamini, T.M., and Polk, H.C. Jr. 1997. Phagocytosis and oxidative burst response of planktonic Staphylococcus epidermidis RP62A and its non‐slime‐prouding variant in human neutrophils. Clin. Diagn. Lab. Immunol. 4:705‐710.
   Huynh, K.K. and Grinstein, S. 2007. Regulation of vacuolar pH and ts modulation by some microbial species. Microbiol. Mol. Biol. Rev. 71:452‐462.
   Kaur, I., Simons, E.R., Castro, V.A., Ott, C.M., and Pierson, D.L. 2004. Changes in neutrophil functions in astronauts. Brain Behav. Immun. 18:443‐450.
   Kaur, I., Simons, E.R., Castro, V.A., Ott, C.M., and Pierson, D.L. 2005. Changes in monocyte functions of astronauts. Brain Behav. Immun. 19:547‐554.
   Kaur, I., Simons, E.R., Kapadia, A.S., Ott, C.M., and Pierson, D.L. 2008. Effect of spaceflight on ability of monocytes to respond to endotoxins of gram‐negative bacteria. Clin. Vaccine Immunol. 15:1523‐1528.
   Krajewski, A., Garg, M., De, M., and Chandawarkar, R.Y. 2009. Phagocytosis: Reemerging roles for a primitive function. Plast. Reconstr. Surg. 123:834‐847.
   Levitz, S.M., Nong, S.‐H., Seetoo, K.F., Harrison, T.S., Speizer, R.A., and Simons, E.R. 1999. Cryptococcus neoformans resides and thrives in an acidic phagolysosome of human macrophages. Infect. Immun. 67:885‐890.
   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.
   Nathan, C. 2006. Neutrophils and Immunity: Challenges and opportunities. Nat. Rev. Immunol. 6:173‐182.
   Ramachandra, L., Smialek, J.L., Shank, S.S., Convery, M., Boom, W.H., and Harding, C.V. 2005. Phagosomal processing of Mycobacterium tuberculosis antigen 85B is modulated independently of mycobacterial viability and phagosome maturation. Infect. Immun. 73:1097‐1105.
   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.
   Steinberg, B.E. and Grinstein, S. 2009. Analysis of macrophage phagocytosis: Quantitative assays of phagosome formation and maturation using high throughput fluorescence microscopy. Methods Mol. Biol. 531:45‐56.
   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.
   Underhill, D.M. and Ozinsky, A. 2002. Phagocytosis of microbes: Complexity in action. Ann. Rev. Immunol. 20:825‐852.
   Voyich, J.M., Braughton, K.R., Sturdevant, D.E., Whitney, A.R., Sais‐Salin, B., Porcella, S.F., Long, D., Dorward, D.W., Gerdner, D.J., Kreiswirth, B.N., Musser, J.M., and DeLeo, F.R. 2005. Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils. J. Immunol. 175:3907‐3919.
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