Measurements of Phagocytosis and Phagosomal Maturation

Chung‐Wai Chow1, Gregory P. Downey1, Sergio Grinstein2

1 University of Toronto and Toronto General Hospital, Toronto, 2 University of Toronto, Toronto, Canada
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.7
DOI:  10.1002/0471143030.cb1507s22
Online Posting Date:  May, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Phagocytosis is an essential component of the innate immune response. Invading microorganisms are engulfed by neutrophils and macrophages, and subsequently killed by a complex series of reactions that require fusion of the phagocytic vacuole with multiple endomembrane organelles. This vacuolar remodeling process, known as phagosomal maturation, is often the target of intracellular parasites that evade killing by the host immune system. This unit describes detailed protocols for the assessment of phagosome formation and maturation. Particular attention is given to Fc‐ and complement‐receptor mediated phagocytosis, but the protocols provided can be readily adapted to other types of phagocytic processes.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Assessment of Fcγ Receptor‐Mediated Phagocytosis
  • Basic Protocol 1: Assessment of FcγR‐Mediated Phagocytosis Using IgG‐Opsonized Red Blood Cells (RBCs)
  • Alternate Protocol 1: Assessment of FcγR‐Mediated Phagocytosis Using IgG‐Opsonized Latex Particles
  • Support Protocol 1: Assessment of Surface Expression and Binding Capacity of Fcγ
  • Assessment of Complement‐Mediated Phagocytosis
  • Basic Protocol 2: Assessment of Complement‐Mediated Phagocytosis Using C3bi‐Opsonized Erythrocytes
  • Alternate Protocol 2: Assessment of Complement‐Mediated Phagocytosis Using C3bi‐Opsonized Latex Beads
  • Alternate Protocol 3: C3bi Opsonization of Beads
  • Basic Protocol 3: Assessment of Phagocytosis Using Flow Cytometry
  • Determination of Phagosomal pH
  • Basic Protocol 4: Measuring Phagosomal pH with Permeant Reagent
  • Alternate Protocol 4: Quantitative pH Determinations by Fluorescence Ratio Microscopy
  • Support Protocol 2: Ratio Imaging Setup
  • Assessment of Phagosomal Maturation
  • Basic Protocol 5: Assessment of the Presence of Transferrin Receptors in Early Phagosomes
  • Alternate Protocol 5: Assessment of the Delivery of Fluid‐Phase Markers to the Phagosome
  • Alternate Protocol 6: Assessment of Phagosomal Maturation by Immunostaining of Endogenous Organellar Markers
  • Basic Protocol 6: Inhibition of Phagocytosis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Assessment of FcγR‐Mediated Phagocytosis Using IgG‐Opsonized Red Blood Cells (RBCs)

  Materials
  • Primary monocytes or macrophages (unit 2.2), or monocytic cell lines (e.g., RAW 264.7 cells ATCC #TIB‐71 or J774 cells, ATCC #HB‐197).
  • Culture medium: e.g., DMEM with or without 10% FBS ( appendix 2B); nominally bicarbonate‐free, HEPES‐buffered media can be used to obviate the need for a CO 2 atmosphere
  • Sheep red blood cells (sRBCs; 10% v/v suspension, ICN Biomedicals)
  • Phosphate‐buffered saline (PBS; without Ca2+ or Mg2+; see recipe)
  • 4% (v/v) paraformaldehyde in PBS (optional)
  • PBS (see recipe) containing 100 mM glycine
  • Rabbit IgG stock: reconstitute rabbit antibody (IgG fraction) to sRBC (ICN Biomedicals) with Milli‐Q water to 5 mg/ml (store in aliquots at –20°C; store thawed aliquots up to 1 month at 4°C)
  • HEPES‐buffered RPMI 1640 (e.g., Life Technologies)
  • Texas Red–sulfonyl chloride (Molecular Probes, 1 mg per ampule)
  • Methanol, prechilled to –20°C
  • Fluorescence mounting medium (Dako)
  • 6‐well tissue culture plates containing sterile glass coverslips (see recipe)
  • End‐over‐end rotator
  • Glass microscope slides
  • Epifluorescence microscope with bright‐field capabilities and appropriate filters (see units 4.1 & 4.2 and 1.NaN)
  • Additional reagents and equipment for cell culture and counting cells (unit 1.1) and microscopy (units 4.1 & 4.2)

Alternate Protocol 1: Assessment of FcγR‐Mediated Phagocytosis Using IgG‐Opsonized Latex Particles

  • 10% slurry of latex beads, ∼3‐µm diameter (Bangs Laboratories cat no. PS05N or equivalent from other manufacturers)
  • Human IgG stock solution: reconstitute IgG from human serum (Sigma) to 50 mg/ml in PBS (see recipe) or Milli‐Q purified H 2O (store in aliquots at –20°C; store thawed aliquots up to 1 month at 4°C)
  • Fluorescently labeled anti‐human IgG secondary antibody (e.g., Cy3‐ or FITC‐labeled anti‐human IgG F(ab) 2, Jackson Laboratories), diluted 1/500 in PBS (see recipe) containing 1% (w/v) BSA and 5% (v/v) goat serum
  • 15‐ml polystyrene tubes, sterile
  • End‐over‐end rotator
  • Tabletop centrifuge

Support Protocol 1: Assessment of Surface Expression and Binding Capacity of Fcγ

  • FcγR‐specific antibody: e.g., rat anti‐mouse CD 16/CD32 (BD Bioscience)
  • Fluorescently labeled secondary antibody (Jackson Immunoresearch Labs)

Basic Protocol 2: Assessment of Complement‐Mediated Phagocytosis Using C3bi‐Opsonized Erythrocytes

  Materials
  • Primary monocytes or macrophages (unit 2.2) or monocytic cell lines (e.g., RAW 264.7 cells ATCC #TIB‐71 or J774 cells, ATCC #HB‐197)
  • Culture medium: e.g., DMEM with or without 10% FBS ( appendix 2B); nominally bicarbonate‐free, HEPES‐buffered media can be used to obviate the need for a CO 2 atmosphere
  • Sheep red blood cells (sRBCs; 10% v/v suspension, ICN Biomedicals)
  • Phosphate‐buffered saline (without Ca2+ or Mg2+; see recipe)
  • Gelatin veronal buffer (GVB; Sigma)
  • Rabbit anti‐sheep erythrocyte IgM (Cedarlane or Accurate Chemical): reconstitute lyophilized antibody in 1 ml H 2O (final concentration 1 mg/ml) and store aliquots at –20°C (avoid repeated freeze‐thaw cycles)
  • C5‐deficient human serum (Sigma)
  • GVB (Sigma) supplemented with 1% (w/v) BSA and 7.5% (v/v) goat serum (optional)
  • Goat anti‐C3bi antibody (optional; Sigma)
  • GVB (Sigma) supplemented with 5% (w/v) BSA and 7.5% (v/v) donkey serum (optional)
  • FITC‐conjugated donkey anti‐goat IgG (Jackson ImmunoResearch Labs; optional)
  • HEPES‐buffered DMEM (e.g., Life Technologies)
  • 20 mM phorbol 12‐myristate 13‐acetate (PMA) in DMSO
  • Methanol, prechilled to –20°C
  • Fluorescence mounting medium (Dako)
  • 6‐well tissue culture plates containing sterile glass coverslips (see recipe)
  • End‐over‐end rotator
  • Glass microscope slides
  • Epifluorescence microscope with bright‐field capabilities and appropriate filters to visualize FITC fluorescence (see units 4.1 & 4.2 and 1.NaN)
  • Additional reagents and equipment for cell culture and counting cells (unit 1.1) and microscopy (units 4.1 & 4.2)

Alternate Protocol 2: Assessment of Complement‐Mediated Phagocytosis Using C3bi‐Opsonized Latex Beads

  • 10% slurry of latex beads, ∼3‐µm diameter (Bangs Laboratories cat no. PS05N or equivalent from other manufacturers)
  • 1 mg/ml human IgM stock (Sigma; store in aliquots at –20°C)
  • Human or mouse serum, freshly isolated, diluted 1:1 in PBS (see recipe for PBS)
  • Hanks' balanced salt solution (HBSS; see recipe)
  • 20 mM phorbol 12‐myristate 13‐acetate (PMA) in DMSO
  • Goat anti‐human C3bi antibody (Sigma)
  • PBS (see recipe) supplemented with 1% (w/v) BSA and 7.5% (v/v) goat serum (optional)
  • FITC‐conjugated donkey anti‐goat IgG (optional)
  • 4% (v/v) paraformaldehyde in PBS (optional)
  • PBS (see recipe) supplemented with 1% (w/v) BSA and 7.5% (v/v) donkey serum (optional)
  • PBS (see recipe) containing 100 mM glycine (optional)
  • 15‐ml polystyrene tubes, sterile

Alternate Protocol 3: C3bi Opsonization of Beads

  Materials
  • Zymosan A (from S. cerevisiae; Sigma)
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.4
  • 0.1 M sodium carbonate, pH 9.3
  • 1 mg/ml solution of reactive fluorophore, e.g., fluorescein isothiocyanate (FITC) or Oregon Green 514 succinimidyl ester, in 0.1 M sodium carbonate, pH 9.3
  • Bacteria of interest as phagocytic target (e.g., E. coli K‐12 strain)
  • 1 mg/ml fluorescein isothiocyanate (FITC) in PBS (see recipe), pH 8.0
  • 1% (v/v) glutaraldehyde in PBS (see recipe for PBS)
  • PBS (see recipe) containing 100 mM glycine
  • Phagocytic cells of interest: cultured cell lines or primary isolates of peripheral blood neutrophils or monocytes (e.g., unit 2.2)
  • PBS (see recipe) containing 0.02% EDTA
  • PBS (see recipe) containing 1.25 mg/ml trypan blue
  • 0.25% trypsin/EDTA or nonenzymatic cell dissociation solution (both available from Sigma)
  • Quenching antibody to fluorophore (e.g., anti‐FITC/Oregon Green Cat# A‐889; anti‐Alexa Fluor 488 Cat # A‐11094 Molecular Probes)
  • Bath sonicator (e.g., Bransonic 1200 Ultracleaner, Branson)
  • Rotating shaker
  • Polypropylene tubes for use with flow cytometer
  • 24‐ or 96‐well tissue culture plates (Polypropylene Cluster, Costar)
  • Flow cytometer
  • Additional reagents and equipment for flow cytometry (e.g., Robinson et al., )

Basic Protocol 3: Assessment of Phagocytosis Using Flow Cytometry

  Materials
  • Cells of interest (e.g., RAW264.7 macrophages; ATCC #TIB‐71)
  • Na+‐rich medium (see recipe), 37°C
  • Fluorophore‐coupled phagocytic particles (see other protocols in this unit)
  • 1 mM stock of LysoTracker Red DND 99 (Molecular Probes) in DMSO
  • 6‐well tissue culture plates containing sterile glass coverslips (see recipe)
  • Leiden chambers and thermostatted holder for microscope stage
  • Fluorescence imaging microscope system with rhodamine filter set
  • Light microscope with differential interference contrast (DIC) capability (unit 4.1)
  • Additional reagents and equipment for light and fluorescence microscopy (unit 4.1)

Basic Protocol 4: Measuring Phagosomal pH with Permeant Reagent

  • Fluorophore‐coupled zymosan particles (see protocol 7, steps to )
  • K+‐rich medium, pH 7.5 and lower pHs for calibration (see recipe)
  • 1 mg/ml nigericin stock in ethanol
  • Ratio imaging setup (see protocol 10)

Alternate Protocol 4: Quantitative pH Determinations by Fluorescence Ratio Microscopy

  Materials
  • Primary monocytes or macrophages (unit 2.2), or monocytic cell lines (e.g., RAW 264.7 cells, ATCC #TIB‐71 or J774 cells, ATCC #HB‐197)
  • DMEM with 10% FBS ( appendix 2B)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Serum‐free DMEM (e.g., Life Technologies)
  • Fluorophore‐conjugated transferrin (Tfn; Molecular Probes; store in small aliquots at −20°C)
  • HEPES‐buffered DMEM (e.g., Life Technologies)
  • 6‐well tissue culture plates containing sterile glass coverslips (see recipe)
  • Fluorescence microscope with appropriate filters (unit 4.2 and appendix 1E)
  • Additional reagents and equipment for phagocytosis assay using sRBCs (see protocol 1 or protocol 2) or latex beads (see protocol 2 or protocol 52) and fluorescence microscopy (unit 4.2)

Support Protocol 2: Ratio Imaging Setup

  Materials
  • Primary monocytes or macrophages (unit 2.2), or monocytic cell lines (e.g., RAW 264.7 cells, ATCC #TIB‐71 or J774 cells, ATCC #HB‐197)
  • DMEM with 10% FBS ( appendix 2B)
  • Fluorophore‐conjugated dextran, MW 3000, lysine fixable (Molecular Probes; store in dark in small aliquots at −20°C)
  • 6‐well tissue culture plates containing sterile glass coverslips (see recipe)
  • Fluorescence microscope with appropriate filters (unit 4.2 and appendix 1E)
  • Additional reagents and equipment for phagocytosis assay using sRBCs (see protocol 1 or protocol 2) or latex beads (see protocol 2 or protocol 52) and fluorescence microscopy (unit 4.2)

Basic Protocol 5: Assessment of the Presence of Transferrin Receptors in Early Phagosomes

  Materials
  • Permeabilization buffer: e.g., 0.1% (v/v) Triton X‐100 in PBS or 2% (w/v) saponin in PBS (see recipe for PBS)
  • Blocking buffer: PBS (see recipe) containing 1% (w/v) BSA and 7.5% (v/v) of serum in which primary antibody was raised)
  • Primary antibody directed against the protein marker of interest (Table 15.7.1)
  • Fluorophore‐conjugated secondary antibody
  • Fluorescence mounting medium (Dako)
  • Additional reagents and equipment for phagocytosis assay (see protocol 1 or protocol 2) and fluorescence microscopy (unit 4.2)

Alternate Protocol 5: Assessment of the Delivery of Fluid‐Phase Markers to the Phagosome

  Materials
  • 20 to 50 µM wortmannin stock solution in DMSO or 1 to 2 mM cytochalasin D stock solution in ethanol (see appendix 1B for more information on these reagents)
  • Additional reagents and equipment for phagocytosis assay (see protocol 1 or protocol 2)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Aderem, A. and Underhill, D.M. 1999. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17:593‐623.
   Bassoe, C.F. and Solberg, C.O. 1984. Phagocytosis of Staphylococcus aureus by human leukocytes: Quantitation by a flow cytometric and a microbiological method. Acta Pathol. Microbiol. Immunol. Scand. 92:43‐53.
   Demaurex, N. 2002. pH Homeostasis of cellular organelles. News Physiol Sci. 17:1‐5.
   Demaurex, N., Romanek, R., Rotstein, O., and Grinstein, S. 1998. Measurement of cytosolic pH in single cells by dual excitation fluorescence imaging. Simultaneous visualization using differential interference contrast optics. In Cell Biology: A Laboratory Handbook, 2nd edition. (J. Celis, ed.) pp. 380‐386. Academic Press, San Diego.
   Greenberg, S. and Grinstein, S. 2002. Phagocytosis and innate immunity. Curr. Opin. Immunol. 14:136‐45.
   Harvath, L. and Terle, D.A. 1999. Assay for phagocytosis. Methods Mol. Biol. 115:281‐289.
   Heyman, B. 2000. Regulation of antibody responses via antibodies, complement, and Fc receptors. Annu. Rev. Immunol. 18:709‐737.
   Lehmann, A.K., Sornes, S., and Halstensen, A. 2000. Phagocytosis: Measurement by flow cytometry. J. Immunol. Methods 243:229.
   McKenzie, S.E. and Schreiber, A.D., 1998. Fc gamma receptors in phagocytes. Curr. Opin. Hematol. 5:16‐21.
   Newman, S.L. and Mikus, L.K. 1985. Deposition of C3b and iC3b onto particulate activators of the human complement system. Quantitation with monoclonal antibodies to human C3. J. Exp. Med. 161:1414‐1431.
   Newman, S.L., Becker, S., and Halme, J. 1985. Phagocytosis by receptors for C3b (CR1), iC3b (CR3), and IgG (Fc) on human peritoneal macrophages. J. Leukoc. Biol. 38:267‐278.
   Oben, J.A. and Foreman, J.C. 1988. A simple quantitative fluorimetric assay of in vitro phagocytosis in human neutrophils. J. Immunol. Methods 112:99‐103.
   Robinson, J.P., Darzynkiewicz, Z., Dean, P.N., Dressler, L.G., Orfao, A., Rabinovitch, P.S., Stewart, C.S., Tanke, H.J., and Wheeless, L.L. (eds.) 2003. Current Protocols in Cytometry. John Wiley & Sons, New York.
   Santos, J.L., Montes, M.J., Gutierrez, F., and Ruiz, C. 1995. Evaluation of phagocytic capacity with a modified flow cytometry technique. Immunol. Lett. 45:1‐14
   Steinkamp, J.A., Wilson, J.S., Saunders, G.C., and Stewart, C.C. 1982. Phagocytosis: Flow cytometric quantitation with fluorescent microspheres. Science 215:64‐67.
   Trinkle, L.S., Wellhausen, S.R., and McLeish, K.R. 1987. A simultaneous flow cytometric measurement of neutrophil phagocytosis and oxidative burst in whole blood. Diagn. Clin. Immunol. 5:62‐68.
   van Eeden, S.F., Klut, M.E., Walker, B.A., and Hogg, J.C. 1999. The use of flow cytometry to measure neutrophil function. J. Immunol. Methods 232:23‐31.
   Vieira, O.V., Botelho, R.J., and Grinstein, S. 2002. Phagosome maturation: Aging gracefully. Biochem. J. 366:689‐704.
   Wright, S.D., Craigmyle, L.S., and Silverstein, S.C. 1983. Fibronectin and serum amyloid P component stimulate C3b‐ and C3bi‐mediated phagocytosis in cultured human monocytes. J. Exp. Med. 158:1338‐134.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library