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Determining Compartment-Specific Metabolic Fluxes

  • Yannic Nonnenmacher
  • Roberta Palorini
  • Karsten Hiller
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1862)

Abstract

In this chapter, we present an experimental protocol for the targeted metabolic profiling of full cells and mitochondria in selectively permeabilized cells. Mitochondria of adherent cell cultures are made accessible by the addition of digitonin—a compound that selectively permeabilizes the cytosolic membrane without affecting mitochondrial integrity. The generated in situ mitochondria are subsequently used in a stable isotope labeling assay in which their metabolic fluxes can be analyzed without any interfering influence originating from cytosolic components. The protocol is complemented by oxygen consumption measurements of permeabilized cells on a Seahorse XF instrument. The additional data on mitochondrial respiration can be used to validate the functionality of mitochondria in the applied setup but are also a valuable add-on to the stable isotope labeling data.

Key words

Stable-isotope Mitochondria Metabolism Metabolic flux Compartmentalization Mass-spectrometry Seahorse XF analyzer 

Notes

Acknowledgments

We would like to thank Prof. Ferdinando Chiaradonna for careful revision of the manuscript.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Yannic Nonnenmacher
    • 1
  • Roberta Palorini
    • 2
  • Karsten Hiller
    • 1
    • 3
  1. 1.Department of Bioinformatics and Biochemistry, Braunschweig Integrated Center of Systems Biology (BRICS)Technische Universität BraunschweigBraunschweigGermany
  2. 2.Department of Biotechnology and BiosciencesUniversity of Milano-BicoccaMilanItaly
  3. 3.Computational Biology of Infection ResearchHelmholtz Centre for Infection ResearchBraunschweigGermany

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