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

Elizabeth R. Simons¹

¹Boston 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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Introduction
Basic Protocol: 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: 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

Figure 9.31.1

pHrodo Bioparticles (Invitrogen/Molecular Probes) were labeled according to the Basic Protocol with OregonGreen SE and AlexaFluor350 SE. The fluorescence of a 1 µg/ml suspension in PBS at various pHs was then measured in a Hitachi 4500 spectrofluorimeter (_exc 565, 488, and 357, _em 590, 530, and 450, respectively). The ratio of F590/F450 and F530/F450 was then calculated. The fluorescence of AlexaFluor350 did not vary detectably over the pH range. Because the ratios were too disparate to permit reasonable comparison, the % of the ratio at each pH was calculated and is plotted here, the ratios at pH 7.68 being arbitrarily defined as 100%.

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Figure 9.31.2

Mycobacterium avium was triple-labeled with AlexaFluor350/pHrodo/DCF, opsonized with autologous serum, and added to PMN at t = 0 according to the Basic Protocol . Fluorescence emission was recorded continuously at 450 nm (Alexafluor 350; panel A), 530 nm (DCF; panel B), or 590 nm (pHrodo; panel C), a shown in the respective panels. Trypan blue (tpb) was added according to the Basic Protocol to distinguish between enclosed (non-quenched) and bound but not enclosed (exposed to trypan blue, therefore quenched) organisms either 15 sec before (15b) or at the indicated times after the Mycobacterium avium suspension (one sec after = 1a, 30 sec after = 30a). The control, i.e., unquenched, fluorescence is indicated as Ma.

View Image

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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	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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