Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry

Gerhard Krumschnabel; Andrea Eigentler; Mario Fasching; Erich Gnaiger

doi:10.1016/B978-0-12-416618-9.00009-1

Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry

Methods Enzymol. 2014:542:163-81. doi: 10.1016/B978-0-12-416618-9.00009-1.

Authors

Gerhard Krumschnabel¹, Andrea Eigentler², Mario Fasching¹, Erich Gnaiger³

Affiliations

¹ OROBOROS INSTRUMENTS, Innsbruck, Austria.
² Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria.
³ OROBOROS INSTRUMENTS, Innsbruck, Austria; Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria. Electronic address: erich.gnaiger@oroboros.at.

PMID: 24862266
DOI: 10.1016/B978-0-12-416618-9.00009-1

Abstract

The mitochondrial transmembrane potential (Δψmt or mtMP) is directly influenced by oxidative phosphorylation (OXPHOS). The exact nature of the interactions between respiration (flux) and mtMP (force) under various physiological and pathological conditions remains unclear, partially due to methodological limitations. Here, we describe a combination of high-resolution respirometry and fluorometry based on the OROBOROS Oxygraph-2k and the widely applied mtMP indicator safranin. The analysis of OXPHOS in mouse brain homogenates revealed that, at commonly applied concentrations, safranin inhibits Complex I-driven OXPHOS capacity, primarily targeting the phosphorylation system, but has no effects on LEAK respiration. Conversely, Complex II-driven OXPHOS capacity was inhibited by <20% by safranin concentrations normally used for mtMP monitoring. The mtMP was higher in the LEAK state without adenylates than at identical LEAK respiration after ADP stimulation and Complex V inhibition with oligomycin. The maximal electron transfer system (ETS) capacity was reached in uncoupler titrations before the mtMP fully collapsed, whereas respiration was inhibited at increasing uncoupler concentrations, resulting in the progressive reduction of mtMP. In a pharmacologically induced state of Complex II dysfunction, mtMP was rather insensitive to the inhibition of OXPHOS to 50% of its normal capacity, but robustly responded to inhibitors when respiration was limited by substrate depletion. The optimal concentration of uncoupler supporting maximal ETS capacity varied as a function of pharmacological intervention. Taken together, the combined measurement of respiration and mtMP greatly enhances the informative potential of OXPHOS studies. The respirometric validation of inhibitory and uncoupling effects is mandatory for any fluorophore employed to assess mtMP in any respiratory state, tissue type, and pathophysiological condition. The methodological issues analyzed herein are relevant for the study of mitochondrial respiration in a wide variety of setting, including cancer cell metabolism.

Keywords: Complex I; Complex II; Electron transfer system; High-resolution respirometry; Mitochondrial membrane potential; OXPHOS analysis; Safranin.

MeSH terms

Animals
Biochemistry / methods*
Cell Respiration
Fluorescent Dyes
Fluorometry / methods*
Male
Membrane Potential, Mitochondrial*
Mice, Inbred C57BL
Oxidative Phosphorylation
Phenazines*

Substances

Fluorescent Dyes
Phenazines
safranine T