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Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells

Nature Protocols volume 7, pages 1068–1085 (2012)Cite this article

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Abstract

Measurements of glycolysis and mitochondrial function are required to quantify energy metabolism in a wide variety of cellular contexts. In human pluripotent stem cells (hPSCs) and their differentiated progeny, this analysis can be challenging because of the unique cell properties, growth conditions and expense required to maintain these cell types. Here we provide protocols for analyzing energy metabolism in hPSCs and their early differentiated progenies that are generally applicable to mature cell types as well. Our approach has revealed distinct energy metabolism profiles used by hPSCs, differentiated cells, a variety of cancer cells and Rho-null cells. The protocols measure or estimate glycolysis on the basis of the extracellular acidification rate, and they measure or estimate oxidative phosphorylation on the basis of the oxygen consumption rate. Assays typically require 3 h after overnight sample preparation. Companion methods are also discussed and provided to aid researchers in developing more sophisticated experimental regimens for extended analyses of cellular bioenergetics.

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Figure 1
Figure 2
Figure 3
Figure 4: OCR, ECAR and OCR/ECAR ratios for hPSCs and human fibroblasts.
Figure 5: Bioenergetic profile of hPSCs and human fibroblasts.
Figure 6: Comparison of data obtained with the XF24 Extracellular Flux Analyzer and conventional energy metabolism profiling methods.
Figure 7: Free fatty acid oxidation in hPSCs and human fibroblasts determined with the XF24 Extracellular Flux Analyzer.
Figure 8: Using the XF24 Extracellular Flux Analyzer with a wide variety of mammalian cell types.

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Acknowledgements

We thank J. Tang for hESCs. Supported by CIRM grants RS1-00313, RB1-01397, TB1-01183, TG2-01169, a training grant from the Broad Stem Cell Research Center at the University of California Los Angeles, and US National Institutes of Health grants GM061721, GM073981, PNEY018228, P01GM081621, CA156674 and CA90571. C.M.K. is an Established Investigator of the American Heart Association and M.A.T. was a Scholar of the Leukemia and Lymphoma Society. We thank D. Wallace (University of Pennsylvania) for Rho0 143B TK− human osteosarcoma cells.

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Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California, USA

    Jin Zhang, Kiyoko Setoguchi, Jason S Hong, Christine M Van Horn & Michael A Teitell

  2. Department of Chemistry and Biochemistry, University of California, Los Angeles, California, USA

    Esther Nuebel & Carla M Koehler

  3. Department of Biology, California State University Northridge, Northridge, California, USA

    Dona R R Wisidagama, Sarah S Imam & Cindy S Malone

  4. Department of Human Genetics, University of California, Los Angeles, California, USA

    Laurent Vergnes

  5. Molecular Biology Institute, University of California, Los Angeles, California, USA

    Carla M Koehler & Michael A Teitell

  6. Broad Stem Cell Research Center, Jonsson Comprehensive Cancer Center, Center for Cell Control, California NanoSystems Institute, University of California, Los Angeles, California, USA

    Michael A Teitell

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Contributions

J.Z.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; E.N.: conception and design, data collection and analysis, manuscript writing; D.R.R.W.: collection and assembly of data, data analysis and interpretation; K.S., J.S.H., C.M.V.H., S.S.I., L.V.: technical support and assistance; C.S.M.: conception and design, data analysis and interpretation, manuscript writing; C.M.K.: conception and design, data analysis and interpretation, manuscript writing; M.A.T.: conception and design, data analysis and interpretation, manuscript writing, final approval of the manuscript and financial support.

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Correspondence to Michael A Teitell.

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Supplementary information

Supplementary Fig. 1

Overview of assessed metabolic pathways. Glucose enters into the glycolytic pathway (orange panel). Pyruvate, a cytosolic product of glycolysis, can be converted into lactate, which can be excreted from cells using monocarboxylate transporters in the plasma membrane. Assessment of the extracellular acidification rate (ECAR) using the XF24 Extracellular Flux Analyzer (indicated by a seahorse icon; Seahorse Bioscience) is a good approximation of lactate excretion under most circumstances. Pyruvate can also be converted into two molecules of acetyl-CoA, which can enter the citric acid cycle (blue circle) within the mitochondrial matrix. The citric acid cycle generates energy equivalents that feed the electron transport chain for cell respiration (green panel). The activity of individual electron transport chain complexes (CI – IV and CV, the ATP synthase) can be assessed using specific inhibitors (indicated next to the complexes they inhibit) by measuring oxygen consumption with either the XF24 analyzer or a Clark-type oxygen electrode. The glycerol-3-phosphate-cycle (purple panel) links the glycolytic pathway, through the intermediate dihydroxyacetone-phosphate, to the electron transport chain. The flavoprotein-dehydrogenase (FP-dehydrogenase) transfers electrons from glycerol-3-phosphate to FAD to generate FADH2 that subsequently passes electrons from ubiquinone (Q) to ubiquinol (QH2). Finally, QH2 supplies electrons to the electron transport chain (see glycerol-3-phosphate as a substrate in Table 3). (TIFF 2070 kb)

Supplementary Fig. 2

OCR/ECAR ratios for normal human dermal fibroblast (NHDF) cells grown with or without 10 µM ROCK inhibitor (RI). Error bars represent the mean ± SD (n = 3). Figure is reprinted with permission from ref 14. (TIFF 547 kb)

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Zhang, J., Nuebel, E., Wisidagama, D. et al. Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells. Nat Protoc 7, 1068–1085 (2012). https://doi.org/10.1038/nprot.2012.048

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