Assessment of Phagocyte Functions by Flow Cytometry

Carl‐Fredrik Bassøe1

1 Haukeland University Hospital, Bergen, Norway
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.19
DOI:  10.1002/0471142956.cy0919s21
Online Posting Date:  August, 2002
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Abstract

Phagocytes neutrophils, monocytes, and macrophages are crucial in the defense against infection. Their functions include phagocytosis, intra‐ and extracellular digestion of targets, oxidative burst, and chemotaxis. This extensive, detailed unit outlines a four‐part procedure for in‐depth investigation of these four functions that reflect the attacking and processing of pathogenic microorganisms. Written by the original instigator of flow‐based assays in this area, this unit defines an area that is as old as flow itself. Numerous support protocols provide preparative procedures for bacterial targets, opsonins, and antigen‐coated beads.

     
 
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Table of Contents

  • Basic Protocol 1: Phagocytosis (Attachment and Internalization) and Phagosomal pH
  • Basic Protocol 2: Processing of Bacteria and Zymosan Particles
  • Basic Protocol 3: Phagocytosis and Oxidative Burst
  • Basic Protocol 4: Chemotaxis
  • Support Protocol 1: Preparation of White Blood Cell Suspension
  • Support Protocol 2: Preparation of Serum from Peripheral Blood
  • Support Protocol 3: Zymosan‐Activated Serum (ZAS) and Preopsonized Zymosan Particles
  • Support Protocol 4: FITC‐Labeling of Bacteria
  • Support Protocol 5: FITC‐Labeling of Zymosan Particles
  • Support Protocol 6: Dilutions of FITC‐Labeled Zymosan A Particles
  • Support Protocol 7: Antigen Coating of Polystyrene Beads
  • Support Protocol 8: Counting Targets
  • Support Protocol 9: Opsonins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Phagocytosis (Attachment and Internalization) and Phagosomal pH

  Materials
  • Targets:
  •  Bacteria S. aureus Cowan III (NCTC 8532; see protocol 8)
  •  Zymosan particles (see protocol 7 and/or protocol 95)
  •  Polychromatic red fluorescence (PC red)‐dyed microspheres, 1‐µm diameter (Fluoresbrite Plain Microspheres, Polysciences) coated with outer membrane vesicles from meningococci (OMV‐beads; see protocol 11)
  • Sørnes's buffer (see recipe)
  • Opsonins (see protocol 13)
  • Dihydrorhodamine 123 (DHR; see recipe)
  • White blood cell suspension (see protocol 5)
  • Dulbecco's phosphate‐buffered saline (DPBS; see recipe) containing 0.02% (w/v) EDTA, ice cold
  • Trypan blue
  • Vindeløv's high‐salt solution (see recipe)
  • Fluorescent beads (e.g., DNA‐Check, Coulter)
  • Flow cytometer with 488‐nm excitation and filters for the detection of green fluorescence (R123, 505 to 545 nm), orange‐red fluorescence (PC red beads, 560 to 590 nm), and red fluorescence (CD14‐PE‐Cy5, 660 to 700 nm)
  • Non‐pyrogenic 96‐well microtiter plates, sterile
  • 12 × 75–mm tubes suitable for use on the flow cytometer

Basic Protocol 2: Processing of Bacteria and Zymosan Particles

  Materials
  • Cells in recipeVindeløv's high‐salt solution from Basic Protocol protocol 1, step
  • Flow cytometer with 488‐nm excitation and filter set for detection of green (FITC) and red (EB) fluorescence

Basic Protocol 3: Phagocytosis and Oxidative Burst

  • CD14‐PE‐Cy5 monoclonal antibody
  • Desired microspheres (see protocol 11), adjusted to a concentration of 2.5 × 108/ml in Sørnes's buffer (see recipe); the initial microsphere/phagocyte (PMN + MΦ) ratio (R) is 20:1.

Basic Protocol 4: Chemotaxis

  Materials
  • White blood cell suspension (see protocol 5)
  • Sørnes's buffer (see recipe)
  • DPBS (see recipe) containing 0.2% (w/v) EDTA
  • 5% zymosan‐activated serum (ZAS; see protocol 7)
  • Paraformaldehyde (optional)
  • 12 × 75–mm tubes
  • 24‐well transwell chemotaxis plates (6.5‐mm diameter, 3‐µm pore size; Corning)
  • 37°C, 5% CO 2 incubator

Support Protocol 1: Preparation of White Blood Cell Suspension

  Materials
  • Lysing solution (see recipe)
  • Dulbecco's phosphate buffered saline with glucose and BSA (DPBS‐GA; see recipe)
  • Vacutainer tubes containing 100 U preservative‐free heparin (unit 9.7)
  • 50‐ml centrifuge tubes, sterile
  • Additional materials for blood collection

Support Protocol 2: Preparation of Serum from Peripheral Blood

  Materials
  • Freshly extracted peripheral blood
  • Red‐top collection tubes (no anticoagulant)
  • 50‐ml centrifuge tubes
  • Additional equipment for venipuncture

Support Protocol 3: Zymosan‐Activated Serum (ZAS) and Preopsonized Zymosan Particles

  Materials
  • Zymosan A particles (Sigma)
  • DPBS (see recipe)
  • Fresh human serum (see protocol 6)
  • Sørnes's buffer (see recipe)
  • 15‐ml plastic centrifuge tube
  • 37°C incubator with rotator

Support Protocol 4: FITC‐Labeling of Bacteria

  Materials
  • Fluorescein isothiocyanate (FITC)
  • 96% and 70% ethanol
  • Heart infusion broth (HIB; Difco)
  • S. aureus Cowan III (NCTC 8532 O/N)
  • Blood agar plates (e.g., Becton Dickinson)
  • 0.9% (w/v) NaCl
  • Sørnes's buffer (see recipe)
  • Spectrophotometer

Support Protocol 5: FITC‐Labeling of Zymosan Particles

  • Zymosan A particles
  • DPBS (see recipe)
  • 10‐ml glass tubes
  • 0.2‐µm filter
  • End‐over‐end rotator, 37°C

Support Protocol 6: Dilutions of FITC‐Labeled Zymosan A Particles

  • Trypan blue
  • 15‐ml polypropylene tubes

Support Protocol 7: Antigen Coating of Polystyrene Beads

  Materials
  • Fluoresbrite PC red 1.0‐µm microspheres (Polysciences)
  • 0.1 M borate buffer, pH 8.5 (0.1 M boric acid; Polysciences)
  • OMV‐beads with and without 2% (w/v) bovine serum albumin, endotoxin‐free (BSA; Roche Diagnostics)
  • Sodium phosphate storage buffer (Polysciences)
  • End‐over‐end rotator

Support Protocol 8: Counting Targets

  Materials
  • Healthy human serum
  • Hypogammaglobulinemic serum
  • Acute‐phase and convalescent serum
  • Sørnes's buffer (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
   Bassøe, C.‐F., Laerum, O.D., Glette, J., Hopen, G., Haneberg, B., and Solberg, C.O. 1983. Simultaneous measurement of phagocytosis and phagosomal pH by flow cytometry: Role of pol‐ymorphonuclear neutrophilic leukocyte granules in phagosome acidification. Cytometry 4:254‐262.
   Bassøe, C.‐F. 1984. Processing of Staphylococcus aureus and zymosan particles by human leukocytes measured by flow cytometry. Cytometry 5:86‐91.
   Bassøe, C.‐F. 2000. Flow cytometric quantitation of phagocytosis in acute myelogenous leukemia. Acta Haematol. 102:163‐171.
   Bassøe, C.‐F., Smith, I., Sørnes, S., Halstensen, A., and Lehmann, A.K. 2000. Concurrent measurement of antigen‐ and antibody‐dependent oxidative burst and phagocytosis in monocytes and neutrophils. Meth. Enzymol. 21:203‐220.
   Cantinieaux, B., Hariga, C., Courtoy, P., Hupin, J., and Fondu, P. 1989. Staphylococcus aureus phagocytosis. A new cytofluorometric method using FITC and paraformaldehyde. J. Immunol. Methods 121:203‐208.
   Hurst, J.K., Albrich, J.M., Green, T.R., Rosen, H., and Klebanoff, S. 1984. Myeloperoxidase‐dependent fluorescein chlorination by stimulated neutrophils. J. Biol. Chem. 259:4812‐4821.
   Lehmann, A.K., Halstensen, A., Holst, J., and Bassøe, C.‐F. 1997. Functional assays for evaluation of serogroup B meningococcal structures as mediators of human opsonophagocytosis. J. Immunol. Methods 200:55‐68.
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