Metabolic characterization of polarized M1 and M2 bone marrow-derived macrophages using real-time extracellular flux analysis

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Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay. Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage’s metabolic phenotype and inflammatory status.

Original languageEnglish
Article numbere53424
Pages (from-to)e53424
JournalJournal of Visualized Experiments
Issue number105
Publication statusPublished - 28 Nov 2015


  • Cytokine
  • Extracellular flux analysis
  • Glycolysis
  • Immunology
  • Immunometabolism
  • Inflammation
  • Innate Immunity
  • Interleukin-4 (IL-4)
  • Issue 105
  • M1 M2 macrophage activation
  • Macrophage activation
  • Macrophage function
  • Mitochondrial (dys)function
  • Oxidative phosphorylation (OXPHOS)
  • Polarization
  • Seahorse

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