Activation of Murine Macrophages

David M. Mosser1, Ricardo Gonçalves2

1 Cell Biology and Molecular Genetics and the Maryland Pathogen Research Institute University of Maryland College Park, Maryland, 2 Department of General Pathology, Institute of Biological Sciences Federal University of Minas Gerais (UFMG), Belo Horizonte
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.2
DOI:  10.1002/0471142735.im1402s111
Online Posting Date:  November, 2015
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Abstract

Our understanding of cell‐mediated immunity (CMI) has revealed the importance of activated macrophages as key immune effector cells. Over the past decade, we have come to realize that macrophages exhibit remarkable plasticity, and different populations of macrophages with distinct physiologies can develop in response to different stimuli. In fact, it is likely that the number of different macrophage populations that can arise may be as diverse as the activating stimuli that induce them. Some of these stimuli can instruct macrophages to kill microbes (classical activation), lay down extracellular matrix components to promote wound healing (alternative activation), or secrete anti‐inflammatory cytokines to terminate inflammation (regulatory macrophages). New ways to biochemically identify these cells have led to a better understanding of the heterogeneity of activated macrophages. As our understanding of the various macrophage populations increases, so does the potential for therapeutic intervention based on targeting specific populations of activated macrophages. © 2015 by John Wiley & Sons, Inc.

Keywords: macrophages; toll‐like receptors; lipopolysaccharide; immune complexes; interferon‐γ; tumor necrosis factor; IL‐4; IL‐10; IL‐12; mannose receptor

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: The Generation of Classically Activated Macrophages (Ca‐MΦ)
  • Alternate Protocol 1: The Generation of Alternatively Activated Macrophages (AA‐MΦ)
  • The Generation of Regulatory Macrophages (R‐MΦ)
  • Alternate Protocol 2: Generation of Regulatory Macrophages Using IgG‐Opsonized Sheep Red Blood Cells
  • Alternate Protocol 3: Generation of Regulatory Macrophages Using Soluble OVA/Anti‐OVA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: The Generation of Classically Activated Macrophages (Ca‐MΦ)

  Materials
  • Murine bone marrow–derived macrophages or resident peritoneal cells (unit 14.1; Zhang et al., )
  • DMEM/F12‐10 medium (see recipe)
  • Murine recombinant interferon γ (IFN‐γ; R&D Systems, cat. no. 485‐MI) in DMEM/F12‐10
  • Serum‐free DMEM/F12 medium (Life Technologies, cat. no. 10565)
  • Lipopolysaccharide (LPS; 10 to 100 ng/ml Ultrapure LPS, E. coli 0111:B4; Invivogen, cat no. tlrl‐3pelps) in DMEM/F12‐10
  • Flat‐bottom 24‐ or 48‐well tissue culture plates (Thermo Scientific Nunc, cat. no. 142485)

Alternate Protocol 1: The Generation of Alternatively Activated Macrophages (AA‐MΦ)

  Additional Materials (also see Basic Protocol)
  • IL‐4: murine recombinant interleukin‐4 (R&D Systems, cat. no. 404‐mL) in DMEM/F12‐10 (see recipe for DMEM/F1‐10)
  • IL‐13: murine recombinant interleukin‐13 (R&D Systems, cat. no. 413‐mL in DMEM/F12‐10 (see recipe for DMEM/F1‐10)

Alternate Protocol 2: Generation of Regulatory Macrophages Using IgG‐Opsonized Sheep Red Blood Cells

  Materials
  • 10% washed sheep red blood cells (SRBC; Lampire Biological Laboratories, cat. no. 7249008; SRBC should be used within 2 weeks of receiving them)
  • Anti‐SRBC IgG (MP Biomedicals, cat. no. 0855806; we typically store anti‐SRBC frozen at a concentration of 33 μg/ml)
  • Dulbecco's phosphate‐buffered saline without Ca or Mg
  • Mini‐rotator (Glas‐Col or equivalent)
  • 24‐well culture plate
  • Additional reagents and equipment for ELISA (unit 2.1; Hornbeck, )

Alternate Protocol 3: Generation of Regulatory Macrophages Using Soluble OVA/Anti‐OVA

  Materials
  • OVA: 1 mg/ml Chicken egg albumin (OVA, Worthington Biochemical Corporation, cat. no. LS003049) in DMEM/F12 medium
  • Anti‐OVA IgG: 4 mg/ml of rabbit polyclonal IgG to chicken egg albumin (Cappel anti‐OVA IgG, MP Biomedicals, cat. no. 0855304) in DMEM/F12 medium
  • Endotoxin removal buffer for protein solution: EndClean (BioVintage, cat. no.18602)
  • DMEM/F12 medium (e.g., Life Technologies, cat. no. 10565)
  • DMEM/F12‐10 medium (see recipe)
  • Lipopolysaccharide (LPS; 10 to 100 ng/ml Ultrapure LPS, E. coli 0111:B4; Invivogen, cat no. tlrl‐3pelps) in DMEM/F12‐10
  • Detoxi‐gel endotoxin‐ removal column (Life Technologies, cat. no. 20339)
  • Mini‐rotator (Glas‐Col or equivalent)
  • Additional reagents and equipment for ELISA (unit 2.1; Hornbeck, )
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Literature Cited

Literature Cited
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