Assessing Changes in Human Natural Killer Cell Metabolism Using the Seahorse Extracellular Flux Analyzer

  1. Traba, Javier
  2. Antón, Olga M.
Libro:
Methods in Molecular Biology

ISSN: 1064-3745,  1940-6029

ISBN: 9781071621592,  9781071621608

Año de publicación: 2022

Páginas: 165-180

Tipo: Capítulo de Libro

DOI: 10.1007/978-1-0716-2160-8_12 GOOGLE SCHOLAR lock_openAcceso abierto editor

Resumen

Natural killer (NK) cells are cytotoxic cells that mediate anti-tumor and anti-viral immunity. The response of NK cells to different cytokines and stimuli may involve cell survival, proliferation, and changes in their cytotoxic function. These responses will be supported by changes in cellular metabolism. Therefore, changes in NK metabolic parameters could somehow predict changes in NK cell function and cytotoxicity. In this chapter, we describe a protocol to measure NK cell metabolism in primary human NK cells by using an extracellular flux analyzer. This machine measures pH and oxygen changes in the medium and allows the study of NK cell glycolysis and mitochondrial respiration in real time with a small number of cells.

Referencias bibliográficas

  • O’Brien KL, Finlay DK (2019) Immunometabolism and natural killer cell responses. Nat Rev Immunol 19(5):282–290. https://doi.org/10.1038/s41577-019-0139-2
  • Brand MD, Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435(2):297–312. https://doi.org/10.1042/BJ20110162
  • Divakaruni AS, Brand MD (2011) The regulation and physiology of mitochondrial proton leak. Physiology 26(3):192–205. https://doi.org/10.1152/physiol.00046.2010
  • Stram AR, Payne RM (2016) Post-translational modifications in mitochondria: protein signaling in the powerhouse. Cell Mol Life Sci 73(21):4063–4073. https://doi.org/10.1007/s00018-016-2280-4
  • Mookerjee SA, Brand MD (2015) Measurement and analysis of extracellular acid production to determine glycolytic rate. J Vis Exp (106):e53464. https://doi.org/10.3791/53464
  • Mookerjee SA, Gerencser AA, Nicholls DG, Brand MD (2017) Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements. J Biol Chem 292(17):7189–7207. https://doi.org/10.1074/jbc.M116.774471
  • Mookerjee SA, Nicholls DG, Brand MD (2016) Determining maximum glycolytic capacity using extracellular flux measurements. PLoS One 11(3):e0152016. https://doi.org/10.1371/journal.pone.0152016
  • Mookerjee SA, Goncalves RLS, Gerencser AA, Nicholls DG, Brand MD (2015) The contributions of respiration and glycolysis to extracellular acid production. Biochim Biophys Acta 1847(2):171–181. https://doi.org/10.1016/j.bbabio.2014.10.005
  • Tognarelli S, Jacobs B, Staiger N, Ullrich E (2016) Flow cytometry-based assay for the monitoring of NK cell functions. J Vis Exp (116). https://doi.org/10.3791/54615
  • Theorell J, Bryceson YT (2016) Analysis of intracellular Ca(2+) mobilization in human NK cell subsets by flow cytometry. Methods Mol Biol 1441:117–130. https://doi.org/10.1007/978-1-4939-3684-7_10
  • Traba J, Waldmann TA, Anton OM (2020) Analysis of human natural killer cell metabolism. J Vis Exp (160). https://doi.org/10.3791/61466
  • Traba J, Miozzo P, Akkaya B, Pierce SK, Akkaya M (2016) An optimized protocol to analyze glycolysis and mitochondrial respiration in lymphocytes. J Vis Exp (117). https://doi.org/10.3791/54918