AMPK Methods and Protocols

Chapter 18

Assessing Mitochondrial Bioenergetics by Respirometry

in Cells or Isolated Organelles

Guillaume Vial and Bruno Guigas

Abstract

Mitochondrial oxidative phosphorylation is central for generating ATP and maintaining energy homeosta-
sis in most eukaryotic cells. The ex vivo measurement of mitochondrial oxygen consumption rates in intact
cells or isolated organelles is a valuable approach to assess mitochondrial bioenergetics in various experi-
mental conditions. In this chapter, we describe several step-by-step protocols for measuring mitochondrial
respiration in intact cells, permeabilized cells (in situ mitochondria), and isolated organelles using both
Clark-type polarographic oxygen electrode devices and the newly developed oxygen-sensing fluorophore-
based Seahorse technology.

Key wordsMitochondria, Oxidative phosphorylation, Oxygraph, Oxygen consumption, Respiratory

rate, Bioenergetics, Clark electrode, Seahorse, AMPK

1 Introduction

Mitochondria plays a central role in most of the eukaryotic cells by

generating ATP and being involved in maintenance of cellular

energy homeostasis. Oxidative phosphorylation (OXPHOS) is a

set of bioenergetic reactions that take place within the mitochon-

drial inner membrane and couples the oxidation of substrates to the

synthesis of ATP from ADP and inorganic phosphate (Pi)

[1]. According to the widely accepted Mitchell’s chemiosmotic

theory of energy transduction [2], electrons generated by substrate

oxidation are transferred by reduced electron carriers, such as

NADH and FADH 2 , to the mitochondrial electron transport

chain (ETC). The subsequent electron flow through the ETC,

i.e. from the respiratory-chain complex I (NADH) or complex

2 (FADH 2 ) to the respiratory-chain complex IV (cytochrome oxi-

dase), leads to reduction of molecular oxygen, the final electron

acceptor, to form water [1]. The electron transfer flavoprotein-

ubiquinone oxidoreductase, localized at the matrix side of the

mitochondrial inner membrane, can also act as electron acceptor

Dietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_18,©Springer Science+Business Media, LLC 2018

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