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Measuring the Nutrient Metabolism of Adherent Cells in Culture

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1862))

Abstract

Metabolite extraction from cells cultured in vitro enables the comprehensive measurement of intracellular metabolites. These extracts can be analyzed using techniques such as liquid chromatography-mass spectrometry (LC-MS). This chapter describes in detail a method for metabolite extraction from cultured adherent mammalian cells to collect both polar and nonpolar intracellular metabolites. This chapter also describes experimental design considerations for performing stable isotope labeling experiments, and the use of chemical derivatization to increase the number of compounds that can be detected using one chromatography method.

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References

  1. Weibel KE, Mor J-R, Fiechter A (1974) Rapid sampling of yeast cells and automated assays of adenylate, citrate, pyruvate and glucose-6-phosphate pools. Anal Biochem 58(1):208–216.

    Article  CAS  Google Scholar 

  2. de Koning W, van Dam K (1992) A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH. Anal Biochem 204(1):118–123.

    Article  Google Scholar 

  3. Sellick CA, Knight D, Croxford AS et al (2010) Evaluation of extraction processes for intracellular metabolite profiling of mammalian cells: matching extraction approaches to cell type and metabolite targets. Metabolomics 6(3):427–438.

    Article  CAS  Google Scholar 

  4. Lunt SY, Muralidhar V, Hosios AM et al (2015) Pyruvate kinase isoform expression alters nucleotide synthesis to impact cell proliferation. Mol Cell 57(1):95–107.

    Article  CAS  Google Scholar 

  5. Morgan HP, O’Reilly FJ, Wear MA et al (2013) M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation. Proc Natl Acad Sci U S A 110(15):5881–5886.

    Article  CAS  Google Scholar 

  6. Israelsen WJ, Vander Heiden MG (2015) Pyruvate kinase: function, regulation and role in cancer. Semin Cell Dev Biol 43:43–51.

    Article  CAS  Google Scholar 

  7. Chaneton B, Hillmann P, Zheng L et al (2012) Serine is a natural ligand and allosteric activator of pyruvate kinase M2. Nature 491(7424):458–462.

    Article  CAS  Google Scholar 

  8. Christofk HR, Vander Heiden MG, Harris MH et al (2008) The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452(7184):230.

    Article  CAS  Google Scholar 

  9. Anastasiou D, Yu Y, Israelsen WJ et al (2012) Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis. Nat Chem Biol 8(10):839–847.

    Article  CAS  Google Scholar 

  10. Metallo CM, Walther JL, Stephanopoulos G (2009) Evaluation of 13C isotopic tracers for metabolic flux analysis in mammalian cells. J Biotechnol 144(3):167–174.

    Article  CAS  Google Scholar 

  11. Buescher JM, Antoniewicz MR, Boros LG et al (2015) A roadmap for interpreting C metabolite labeling patterns from cells. Curr Opin Biotechnol 34C:189–201.

    Article  Google Scholar 

  12. Fang M, Ivanisevic J, Benton HP et al (2015) Thermal degradation of small molecules: a global metabolomic investigation. Anal Chem 87(21):10935–10941.

    Article  CAS  Google Scholar 

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Correspondence to Sophia Y. Lunt .

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Ogrodzinski, M.P., Teoh, S.T., Yu, L., Broadwater, D., Ensink, E., Lunt, S.Y. (2019). Measuring the Nutrient Metabolism of Adherent Cells in Culture. In: Fendt, SM., Lunt, S. (eds) Metabolic Signaling. Methods in Molecular Biology, vol 1862. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8769-6_3

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  • DOI: https://doi.org/10.1007/978-1-4939-8769-6_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8768-9

  • Online ISBN: 978-1-4939-8769-6

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