Measuring Bioenergetics in T Cells Using a Seahorse Extracellular Flux Analyzer

Gerritje J.W. van der Windt1, Chih‐Hao Chang2, Erika L. Pearce3

1 Department of Experimental Immunology, Academic Medical Center, Amsterdam, 2 Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, Missouri, 3 Department of Immunometabolism, Max Planck Institute of Immunobiology and Epigenetics, Freiburg
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.16B
DOI:  10.1002/0471142735.im0316bs113
Online Posting Date:  April, 2016
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This unit contains several protocols to determine the energy utilization of T cells in real‐time using a Seahorse Extracellular Flux Analyzer ( The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and mitochondrial respiration, in real‐time, on relatively small numbers of cells, without any radioactivity. The Basic Protocol describes a standard mitochondrial stress test on the XFe96, which yields information about oxidative phosphorylation and glycolysis, two energy‐generating pathways. The alternate protocols provide examples of adaptations to the Basic Protocol, including adjustments for the use of the XFe24. A protocol for real‐time bioenergetic responses to T cell activation allows for the analysis of immediate metabolic changes after T cell receptor stimulation. Specific substrate utilization can be determined by the use of differential assay media, or the injection of drugs that specifically affect certain metabolic processes. Accurate cell numbers, purity, and viability are critical to obtain reliable results. © 2016 by John Wiley & Sons, Inc.

Keywords: t cells; bioenergetics; glycolysis; oxidative phosphorylation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Mitochondrial Stress Test on the XF e 96 EFA
  • Alternate Protocol 1: Mitochondrial Stress Test on the XF e 24 EFA
  • Alternate Protocol 2: Real‐Time Bioenergetic Measurements in Response to Activation
  • Alternate Protocol 3: Substrate Utilization: Galactose
  • Alternate Protocol 4: Substrate Utilization: Long‐Chain Fatty Acids
  • Reagents and Solutions
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Mitochondrial Stress Test on the XF e 96 EFA

  • XFe96 FluxPak including: (Seahorse Bioscience)
  •  Sensor cartridges
  •  Cell culture microplates
  •  Calibrant
  • Poly‐D‐lysine (PDL; Sigma; use at 50 μg/ml)
  • XF medium, pH 7.4 (see recipe)
  • Mitochondria interrogating drugs [i.e., 1 mM oligomycin, 1.5 mM Fluoro‐carbonyl cynade phenylhydrazon (FCCP), 100 μM rotenone, and 1 mM antimycin A; Sigma]
  • Defined murine T cell subset suspension (cultured or freshly isolated) in XF medium (see Chapter 3)
  • XFe96 extracellular flux analyzer (Seahorse Bioscience)
  • 37°C non‐CO 2 incubator
  • Centrifuge
  • Additional reagents and equipment for preparing T cell suspensions (see Chapter 3) and counting cells ( ; see Strober, )
NOTE: A sensor cartridge is packaged on top of a microplate, and delivered with loading guides for easy drug loading.NOTE: It is recommended to use at least four replicate wells/group for accuracy of the run. However, acquiring repeated results from independent experiments is critical to validate conclusions (also see Background information).NOTE: If cultured cells are assayed, it is important to realize that cell concentration, nutrient availability, and the surface area of the media for gas exchange (also dependent on the depth of the medium) can affect the metabolic phenotype of cells. Keeping these parameters consistent is critical for comparisons between experiments.NOTE: Standard XF medium lacks serum, so cells should not be left in this media for long periods of time. Depending on the question to be answered, serum can be added to the XF medium. It is important to note that in that case drug concentrations, in particular FCCP, may need to be adjusted (see Background information).

Alternate Protocol 1: Mitochondrial Stress Test on the XF e 24 EFA

  Additional Materials (also see protocol 1Basic Protocol 1)
  • XFe24 extracellular flux analyzer (Seahorse Bioscience)
  • XFe24 FluxPak (Seahorse Bioscience)

Alternate Protocol 2: Real‐Time Bioenergetic Measurements in Response to Activation

  Additional Materials (also see protocol 1Basic Protocol)
  • PMA and ionomycin (Sigma)
  • Anti‐CD3/CD28‐coated beads (Gibco)

Alternate Protocol 3: Substrate Utilization: Galactose

  Additional Materials (also see protocol 1Basic Protocol)
  • Glucose XF medium, pH 7.4 (see recipe)
  • Galactose XF medium, pH 7.4 (see recipe)
NOTE: Activated T cells cultured in glucose will engage aerobic glycolysis and OXPHOS; however, T cells cultured in galactose will not engage aerobic glycolysis, and instead will primarily use mitochondrial respiration. If cells are cultured in galactose‐containing medium before being assayed on the Seahorse EFA, it is important to use glucose‐free medium and to dialyze the serum to ensure a complete glucose‐free medium. All cell groups, whether cultured in glucose or galactose, must be cultured in media that contains the same dialyzed serum.

Alternate Protocol 4: Substrate Utilization: Long‐Chain Fatty Acids

  Additional Materials (also see protocol 1Basic Protocol)
  • 20 mM Etomoxir
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Literature Cited

Literature Cited
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