Extracellular flux analysis to monitor glycolytic rates and mitochondrial oxygen consumption

Methods Enzymol. 2014;542:125-49. doi: 10.1016/B978-0-12-416618-9.00007-8.


Evidence accumulating over the past decade has linked alterations in bioenergetic metabolism to the pathogenesis of several diseases, including inflammatory conditions and cancer. However, the mutual relationship between the effector functions and the metabolism of immune cells has begun to emerge only recently. Similar to malignant cells, both innate and adaptive immune cells undergo a metabolic reprogramming that is required for effector functions, de facto underlying the elicitation of a robust immune response. These changes allow immune cells not only to rapidly respond to pathogens or (pre)malignant cells but also to adapt to changing microenvironmental conditions. Targeting the metabolic alterations of malignant cells has been the subject of an intense wave of investigation, resulting in the identification of promising therapeutic strategies. Since the inflammatory milieu and the tumor microenvironment are similar, the metabolism of immune cells and its regulation has recently come under renewed interest as a target for immunotherapy. Here, we describe different tools and techniques to study the bioenergetic metabolism of cultured cells, using immune cells as a model. Our methodological approach relies on an extracellular flux analyzer, an instrument that enables the real-time measurement of the two central pathways used by living cells to generate adenosine triphosphate: glycolysis and oxidative phosphorylation. This instrument and similar technological innovations have transformed the study of cellular metabolism, unveiling its profound impact on various immunologic and oncological disorders.

Keywords: Bioenergetics; Energy metabolism; Glycolysis; Immune cells; Mitochondrial respiration; Oxidative phosphorylation.

MeSH terms

  • Biochemistry / instrumentation
  • Biochemistry / methods*
  • Cells, Cultured
  • Energy Metabolism
  • Extracellular Space / metabolism
  • Glycolysis*
  • Humans
  • Immune System / cytology*
  • Immune System / metabolism
  • Jurkat Cells
  • Lymphocytes / metabolism
  • Metabolome
  • Mitochondria / metabolism*
  • Myeloid Cells / metabolism
  • Oxidative Phosphorylation
  • Oxygen / metabolism*
  • Tissue Array Analysis


  • Oxygen