Measurement of Phagocytosis, Phagosome Acidification, and Intracellular Killing of Staphylococcus aureus

Anna Sokolovska1, Christine E. Becker1, Lynda M. Stuart2

1 Developmental Immunology/CCIB Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts, 2 The Broad Institute of MIT and Harvard, Cambridge, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.30
DOI:  10.1002/0471142735.im1430s99
Online Posting Date:  November, 2012
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Abstract

Phagocytes are an important part of host defense, playing a critical role in innate immune responses against pathogens and in the initiation of adaptive immunity. One of the main characteristics of these cells is their ability to recognize and internalize invading microorganisms into a phagosome. The internalized microbe is rapidly delivered into a mature phagolysosome where it is killed and degraded. However, numerous pathogens have evolved complex mechanisms to manipulate these intracellular organelles to establish a survival niche. Here, we describe several methods to assess important properties of phagosomes in macrophages, such as phagocytosis, acidification of the phagosome contents during the maturation process, and the ability of phagosomes to inactivate and kill pathogens. Phagocytosis and phagosome acidification assays are FACS‐based assays where labeled bacteria are used as probes to monitor internalization into a phagosome and to detect the pH of the phagosome environment. The killing assay is based on the counting of bacterial colonies after recovery of internalized bacteria from macrophages. Curr. Protoc. Immunol. 99:14.30.1‐14.30.12. © 2012 by John Wiley & Sons, Inc.

Keywords: phagocytosis; phagosome acidification; macrophages; microbial killing; gentamicin protection assay

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Phagocytosis
  • Basic Protocol 2: Measurement of Phagosome Acidification
  • Basic Protocol 3: Gentamicin Protection Assay
  • Support Protocol 1: Bacteria Labeling
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of Phagocytosis

  Materials
  • Primary macrophage cultures or bone marrow–derived macrophages (unit 14.1), or macrophage cell lines
  • DMEM with 10% fetal bovine serum (FBS), with 1× penicillin/streptomycin (also see appendix 2A)
  • Dulbecco's phosphate‐buffered saline (DPBS; e.g., Invitrogen)
  • DMEM with 1% fetal bovine serum (FBS), with and without 1× penicillin/streptomycin (also see appendix 2A)
  • Inhibitors: e.g., cytochalasin D (Sigma, cat. no. C8273) or other inhibiting agents of interest
  • Dimethylsulfoxide (DMSO)
  • S. aureus (live or heat inactivated) or other bacteria or beads labeled with Alexa Fluor 647 fluorescent dyes (see protocol 4)
  • DPBS/EDTA: Dulbecco's phosphate‐buffered saline (DPBS; e.g., Invitrogen) containing 5 mM EDTA
  • 24‐well flat‐bottom tissue culture plates (e.g., Falcon or Costar)
  • 1‐ml syringes with 30‐G needles
  • FACS tubes
  • Additional reagents and equipment for preparing mouse peritoneal macrophages or bone marrow–derived macrophages (unit 14.1) and flow cytometry (unit 5.4)
NOTE: All reagents and equipment coming into contact with live cells until step 5 must be sterile.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Measurement of Phagosome Acidification

  Materials
  • Primary macrophage cultures or bone marrow‐derived macrophages (unit 14.1), or macrophage cell lines
  • DMEM with 10% fetal bovine serum (FBS), with 1× penicillin/streptomycin (also see appendix 2A)
  • Dulbecco's phosphate‐buffered saline (DPBS; e.g., Invitrogen)
  • Inhibitors: e.g., bafilomycin A (Sigma, cat. no. B1793) or other inhibiting agents of interest
  • Dimethylsulfoxide (DMSO)
  • DMEM with 1% fetal bovine serum (FBS), with and without 1× penicillin/streptomycin (also see appendix 2A)
  • S. aureus (live or heat inactivated) or other bacteria or beads labeled with Alexa Fluor 647 fluorescent dyes (see protocol 4)
  • DPBS/EDTA: Dulbecco's phosphate‐buffered saline (DPBS; e.g., Invitrogen) containing 5 mM EDTA
  • pH buffers ( e.g., DPBS titrated with NaOH or NaCl to produce buffers with fixed pH from 3.5 to 9 with 0.5 pH difference) containing 0.05% Triton X‐100.
  • 24‐well flat‐bottom tissue culture plates (e.g., Falcon or Costar)
  • 1‐ml syringes with 30‐G needles
  • FACS tubes
  • Additional reagents and equipment for preparing mouse peritoneal macrophages or bone marrow‐derived macrophages (unit 14.1) and flow cytometry (unit 5.4)
NOTE: All reagents and equipment coming into contact with live cells until step 5 must be sterile.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified

Basic Protocol 3: Gentamicin Protection Assay

  Materials
  • Primary macrophage cultures or bone marrow‐derived macrophages (unit 14.1), or macrophage cell lines
  • DMEM with 10% fetal bovine serum (FBS), with and without 1× penicillin/streptomycin (also see appendix 2A)
  • Live S. aureus (see protocol 4)
  • Dulbecco's phosphate‐buffered saline (DPBS; e.g., Invitrogen)
  • DMEM with 1% fetal bovine serum (FBS), without antibiotics (also see appendix 2A)
  • Gentamicin (Invitrogen)
  • 0.02% (v/v) Triton X‐100 in DPBS
  • Blood agar plates (Remel)
  • 24‐well flat‐bottom tissue culture plates (e.g., Falcon or Costar)
  • 30‐G needles
  • 1‐ml syringes
  • Centrifuge
  • Additional reagents and equipment for preparing mouse peritoneal macrophages or bone marrow‐derived macrophages (unit 14.1)

Support Protocol 1: Bacteria Labeling

  Materials
  • Stock bacterial culture (e.g., S. aureus; other bacteria can be used as well)
  • Columbia broth (BD Difco) supplemented with 2% (w/v) NaCl
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Alexa Fluor 647 carboxylic acid, succinimidyl ester (Invitrogen) or fluorescein‐5‐isothiocyanate (FITC “Isomer I”; Invitrogen)
  • Dimethylsulfoxide (DMSO)
  • Blood agar plates (Remel)
  • Bovine serum albumin (BSA; Sigma)
  • Spectrophotometer
  • GS‐6 Centrifuge (Beckman), refrigerated
  • 14‐ml polystyrene round‐bottom tubes (Falcon)
  • 15‐ml centrifuge tube (Corning)
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Figures

Videos

Literature Cited

Literature Cited
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