Dynamic Quantitative Assays of Phagosomal Function

Maria Podinovskaia1, Brian C. VanderVen1, Robin M. Yates2, Sarah Glennie3, Duncan Fullerton4, Henry C. Mwandumba5, David G. Russell1

1 Cornell University, Ithaca, New York, 2 Faculty of Veterinary Medicine, Calgary, Alberta, 3 Liverpool School of Tropical Medicine, Liverpool, 4 Mid‐Cheshire NHS Foundation Trust, Crewe, 5 Queen Elizabeth Hospital, Chichiri, Blantyre
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.34
DOI:  10.1002/0471142735.im1434s102
Online Posting Date:  October, 2013
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Abstract

Much of the activity of the macrophage as an effector cell is performed within its phagocytic compartment. This ranges from the degradation of tissue debris as part of its homeostatic function to the generation of the superoxide burst as part of its microbicidal response to infection. We have developed a range of real‐time readouts of phagosomal function that enable these activities to be rigorously quantified. This unit contains descriptions of several of these assays assessed by different methods of quantitation, including a fluorescence resonance emission transfer (FRET) assay for phagosome/lysosome fusion measured by spectrofluorometry, a fluorogenic assay for the superoxide burst measured by flow cytometry, and a fluorogenic assay for bulk proteolysis measured by confocal microscopy. These assays illustrate both the range of parameters that can be quantified and the flexibility of instrumentation that can be exploited for their quantitation. Curr. Protoc. Immunol. 102:14.34.1‐14.34.14. © 2013 by John Wiley & Sons, Inc.

Keywords: macrophage; phagosome; phagocytosis; phagosome maturation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Measurement of Phagosome/Lysosome Fusion
  • Basic Protocol 2: Intraphagosomal Measurement of the Magnitude and Duration of the Superoxide Burst
  • Basic Protocol 3: Measurement of the Extent of Proteolytic Activity in Phagosomes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of Phagosome/Lysosome Fusion

  Materials
  • Mice
  • Mø medium (see recipe)
  • Phosphate‐buffered saline (PBS; Invitrogen; also see appendix 2A), pH 7.2 (ice cold)
  • Acceptor fluorochrome: Alexa Fluor 594 hydrazide, sodium salt (Alexa 594‐HA; Molecular Probes)
  • Donor fluorochrome (Alexa 488‐SE)–derivatized, IgG‐opsonized FRET reporter particles consisting of 3.0 µm carboxylate‐modified silica particles (Si‐COOH) (Kisker Biotech, http://www.kisker‐biotech.com/; for preparation, see recipe in Reagents and Solutions)
  • Cuvette buffer (see recipe)
  • 90‐mm‐diameter petri dishes (Kord‐Valmark, cat. no. 2900, http://www.kord‐valmark.com)
  • Sterile 13 × 25– mm glass coverslips (Knittel Glaser, http://www.knittelglass.com/)
  • Rubber policeman (Sarstedt)
  • 4‐chamber petri dishes, sterile (Fisher, cat no. 08‐758‐2)
  • Forceps, sterile
  • 6‐well plates or 35‐mm Ibidi µ‐Dishes (http://ibidi.com/)
  • 50‐ml conical tubes (BD Falcon)
  • Spectrofluorometer (PTI QMSE4; PerkinElmer)
  • Quartz cuvettes
  • Additional reagents and equipment for preparation of macrophages from mice (unit 14.1) and test of cell viability by trypan blue exclusion ( appendix 3B)

Basic Protocol 2: Intraphagosomal Measurement of the Magnitude and Duration of the Superoxide Burst

  Materials
  • Bone‐marrow–derived murine macrophages (Mø; see protocol 1)
  • Mø medium (see recipe)
  • Reporter (Oxyburst Green, succinimidyl ester, H 2DCFDA‐SE; Molecular Probes) and calibration (Alexa Fluor 633 carboxylic acid, succinimidyl ester, Alexa 633‐SE; Molecular Probes) fluorochrome–derivatized, IgG‐opsonized Superoxide Burst reporter particles consisting of 3.0 µm carboxylate‐modified silica particles (Si‐COOH) (Kisker Biotech, http://www.kisker‐biotech.com/; for preparation, see recipe in Reagents and Solutions)
  • Phosphate‐buffered saline (PBS; Invitrogen; also see appendix 2A), ice cold
  • 1% (w/v) paraformaldehyde in PBS
  • 6‐well culture plates
  • Cell scraper
  • Fluorescence activated cell sorter (FACS) or flow cytometer (also see Chapter )
  • Additional reagents and equipment for flow cytometry (Chapter )

Basic Protocol 3: Measurement of the Extent of Proteolytic Activity in Phagosomes

  Materials
  • Bone marrow–derived murine macrophages (Mø; see protocol 1)
  • Mø medium (see recipe)
  • Cuvette buffer (see recipe)
  • Reporter (DQ Green‐BSA; Molecular Probes) and calibrator (Alexa Fluor 633 carboxylic acid, succinimidyl ester, Alexa 633‐SE; Molecular Probes) fluorochrome–derivatized, IgG‐opsonized reporter consisting of 3.0 µm carboxylate‐modified silica particles (Si‐COOH) (Kisker Biotech, http://www.kisker‐biotech.com/; for preparation, see recipe in Reagents and Solutions)
  • Chamber dishes: 35 mm µ‐Dishes ( Ibidi, http://ibidi.com/)
  • Confocal microscope: the microscope should have a stage‐heating system and the chamber should be pre‐heated to 37°C; set up with excitation wavelengths of 488 and 633 nm, and emission range of 505 to 535 nm and 643 to 673 nm, for reporter and calibration fluorophore, respectively; we use a Leica SP5 confocal laser‐scanning system with an inverted microscope
  • Leica Application Suite Advanced Fluorescence (LAS‐AF) software (Leica Microsystems GmbH) for image acquisition and analysis
  • Quantification software: Volocity image analysis software (PerkinElmer Life Sciences) is used for reporter particle tracking and quantification
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Figures

Videos

Literature Cited

Literature Cited
  Blander, J.M. and Medzhitov, R. 2004. Regulation of phagosome maturation by signals from toll‐like receptors. Science 304:1014‐1018.
  Podinovskaia, M., Lee, W., Caldwell, S., and Russell, D.G. 2013. Infection of macrophages with Mycobacterium tuberculosis induces global modifications to phagosomal function. Cell Microbiol. 15:843‐859.
  Russell, D.G. and Yates, R.M. 2007. Toll‐like receptors and phagosome maturation. Nat. Immunol. 8:217‐218.
  Russell, D.G., Vanderven, B.C., Glennie, S., Mwandumba, H., and Heyderman, R.S. 2009. The macrophage marches on its phagosome: Dynamic assays of phagosome function. Nat. Rev. Immunol. 9:594‐600.
  Rybicka, J.M., Balce, D.R., Chaudhuri, S., Allan, E.R., and Yates, R.M. 2012. Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH‐independent manner. EMBO J. 31:932‐944.
  VanderVen, B.C., Yates, R.M., and Russell, D.G. 2009. Intraphagosomal measurement of the magnitude and duration of the oxidative burst. Traffic 10:372‐378.
  VanderVen, B.C., Hermetter, A., Huang, A., Maxfield, F.R., Russell, D.G., and Yates, RM. 2010. Development of a novel, cell‐based chemical screen to identify inhibitors of intraphagosomal lipolysis in macrophages. Cytometry A 77:751‐760.
  Yates, R.M. and Russell, D.G. 2005. Phagosome maturation proceeds independently of stimulation of toll‐like receptors 2 and 4. Immunity 23:409‐417.
  Yates, R.M. and Russell, D.G. 2008. Real‐time spectrofluorometric assays for the lumenal environment of the maturing phagosome. Methods Mol. Biol. 445:311‐325.
  Yates, R.M., Hermetter, A., and Russell, D.G. 2005. The kinetics of phagosome maturation as a function of phagosome/lysosome fusion and acquisition of hydrolytic activity. Traffic 6:413‐420.
  Yates, R.M., Hermetter, A., Taylor, G.A., and Russell, D.G. 2007. Macrophage activation downregulates the degradative capacity of the phagosome. Traffic 8:241‐250.
  Yates, R.M., Hermetter, A., and Russell, D.G. 2009. Recording phagosome maturation through the real‐time, spectrofluorometric measurement of hydrolytic activities. Methods Mol. Biol. 531:157‐171.
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