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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hiller K, Metallo CM (2013) Profiling metabolic networks to study cancer metabolism. Curr Opin Biotechnol 24:60–68
Gravel S-P, Andrzejewski S, Avizonis D et al (2014) Stable isotope tracer analysis in isolated mitochondria from mammalian systems. Metabolites 4:166–183
Chen WW, Freinkman E, Wang T et al (2016) Absolute quantification of matrix metabolites reveals the dynamics of mitochondrial metabolism. Cell 166:1324–1337.e11
Rogers GW, Brand MD, Petrosyan S et al (2011) High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PLoS One 6:e21746
Nonnenmacher Y, Palorini R, d’Herouël AF et al (2017) Analysis of mitochondrial metabolism in situ: combining stable isotope labeling with selective permeabilization. Metab Eng 43:147–155
Battello N, Zimmer AD, Goebel C et al (2016) The role of HIF-1 in oncostatin M-dependent metabolic reprogramming of hepatic cells. Cancer Metab 4:3
Hiller K, Hangebrauk J, Jäger C et al (2009) MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted GC/MS based metabolome analysis. Anal Chem 81:3429–3439
Salabei JK, Gibb AA, Hill BG (2014) Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis. Nat Protoc 9:421–438
Ye F, Hoppel CL (2013) Measuring oxidative phosphorylation in human skin fibroblasts. Anal Biochem 437:52–58
Divakaruni AS, Wiley SE, Rogers GW et al (2013) Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier. Proc Natl Acad Sci U S A 110:5422–5427
Clerc P, Polster BM (2012) Investigation of mitochondrial dysfunction by sequential microplate-based respiration measurements from intact and permeabilized neurons. PLoS One 7:e34465
Acknowledgments
We would like to thank Prof. Ferdinando Chiaradonna for careful revision of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Nonnenmacher, Y., Palorini, R., Hiller, K. (2019). Determining Compartment-Specific Metabolic Fluxes. 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_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8769-6_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8768-9
Online ISBN: 978-1-4939-8769-6
eBook Packages: Springer Protocols