Measuring Autophagy in Macrophages

James Harris1, Orla Hanrahan1, Sergio A. De Haro2

1 School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland, 2 University of New Mexico School of Medicine, Albuquerque, New Mexico
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.14
DOI:  10.1002/0471142735.im1414s87
Online Posting Date:  November, 2009
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Macroautophagy is a conserved intracellular homeostatic mechanism for the degradation of cytosolic constituents. Autophagy can promote cell survival by providing essential amino acids from the breakdown of macromolecules during periods of nutrient deprivation, and can remove damaged or excess organelles, such as mitochondria and peroxisomes. More recently, autophagy has been shown to play an important role in innate and adaptive immune responses to pathogenic bacteria in macrophages and dendritic cells. This unit presents protocols for the measurement of autophagy in macrophages. Curr. Protoc. Immunol. 87:14.14.1‐14.14.13. © 2009 by John Wiley & Sons, Inc.

Keywords: autophagosome; LC3; monodansylcadaverine; Atg; p62; rapamycin

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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Autophagy in Macrophages
  • Basic Protocol 2: Measuring Formation and Localization of LC3‐Positive Autophagosomes by Immunofluorescence
  • Alternate Protocol 1: Measuring Autophagosome Formation and Localization by Immunofluorescence Using MDC
  • Basic Protocol 3: Measuring Autophagosome Formation by Immunoblot Analysis
  • Support Protocol 1: Preparation of Cell Lysates for Immunoblot Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Induction of Autophagy in Macrophages

  • Macrophages (unit 14.1): e.g., cultured macrophage cell lines, murine bone marrow–derived macrophages (BMDM), human peripheral blood–derived macrophages
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • Appropriate culture medium (e.g., DMEM with 10% FBS, 2 mM L‐glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin for BMDM)
  • Sterile Earle's balanced salt solution (EBSS; e.g., Sigma)
  • 0.5 M rapamycin (Sigma) stock solution in DMSO (store in aliquots at −20°C)
  • Dimethylsulfoxide (DMSO) for controls
  • 1 mg/ml IFN‐γ (species‐specific) stock solution in PBS (106 U/ml, depending on supplier and batch)
  • 1 mg/ml lipopolysaccharide (LPS) stock solution (Alexis Biochemicals)
  • Appropriate tissue culture vessels: e.g., 6‐well tissue culture plates to produce cell lysates for immunoblot analysis or 19‐mm‐diameter coverslips in 12‐well tissue culture plates for immunofluorescence studies
  • 50‐ml polypropylene or polystyrene centrifuge tubes
  • Centrifuge
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Measuring Formation and Localization of LC3‐Positive Autophagosomes by Immunofluorescence

  • Macrophages (unit 14.1): e.g., cultured macrophage cell lines, murine bone marrow–derived macrophages (BMDM), human peripheral blood–derived macrophages
  • Specific cell organelle markers (optional; available from Invitrogen), e.g.:
    • MitoTracker dye
    • CellMask plasma membrane stain
    • Deep Red nuclear stain
  • 2% to 4% (w/v) paraformaldehyde
  • 0.1% (v/v) Triton X‐100 in phosphate‐buffered saline (PBS; appendix 2A)
  • Blocking buffer: PBS ( appendix 2A) containing 1% bovine serum albumin (BSA) and 0.5% goat serum (or serum from other species in which secondary antibody was raised)
  • Primary antibody: rabbit anti–LC3 IgG (Sigma, cat. no. L 8918)
  • Secondary antibody: Alexa Fluor–conjugated goat anti–rabbit IgG (Invitrogen)
  • Aqueous mounting medium (see recipe) or polymerizing mounting medium (e.g., Fluorescent Mounting Medium from Dako)
  • 4′,6‐diamidino‐2‐phenylindole (DAPI)
  • Clear nail polish
  • Glass coverslips, no. 1.5 thickness, 19‐mm diameter
  • 12‐well tissue culture plates
  • Microscope slides, 76 × 26 mm
  • Additional reagents and equipment for induction of autophagy ( protocol 1)

Alternate Protocol 1: Measuring Autophagosome Formation and Localization by Immunofluorescence Using MDC

  • 50 mM monodansylcadaverine (MDC) stock solution in DMSO

Basic Protocol 3: Measuring Autophagosome Formation by Immunoblot Analysis

  • Macrophage cell lysates (see protocol 5)
  • 2× SDS sample (Laemmli) buffer (see recipe)
  • 12% SDS‐PAGE gel (unit 8.4)
  • Running buffer (see recipe)
  • Prestained molecular weight standards
  • Nitrocellulose membrane (also see unit 8.10)
  • TBST (see recipe)
  • Blocking buffer (see recipe)
  • Primary antibodies (Sigma):
    • rabbit anti–LC3 IgG
    • rabbit anti‐p62 antibody
    • mouse anti‐β‐actin monoclonal antibody (can be substituted by antibodies against tubulin, VDAC1/porin, COXIV)
  • Antibody dilution buffer (see recipe)
  • Secondary antibody (e.g., Sigma): goat anti–rabbit IgG conjugated to horseradish peroxidase (HRP) for rabbit primary antibodies; goat anti–mouse IgG conjugated to HRP for mouse primary antibody
  • Chemiluminescent HRP substrate (also see unit 8.10)
  • X‐ray film
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and immunoblotting (unit 8.10)

Support Protocol 1: Preparation of Cell Lysates for Immunoblot Analysis

  • Macrophages (unit 14.1) growing in culture in multiwell plates (typically 6‐ or 12‐well plates, but 24‐well plates can also be used)
  • Phosphate‐buffered saline (PBS; appendix 2A), at 4°C
  • Lysis buffer (see recipe), ice cold
  • Protein assay kit (e.g., for Bradford assay)
  • Cell scrapers
  • Tabletop centrifuge (e.g., IEC Clinical)
  • Microcentrifuge
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Key Reference
   Klionsky et al., 2008 . see above.
  An invaluable article presenting the consensus of experts in the field on the interpretation of autophagy assays, including immunoblot analysis and fluorescent microscopy, as well as electron microscopy and studies of autophagic flux and protein degradation.
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