Krumschnabel 2014 Methods Enzymol
| Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. https://doi.org/10.1016/B978-0-12-416618-9.00009-1 |
» PMID: 24862266 »
Krumschnabel Gerhard, Eigentler Andrea, Fasching Mario, Gnaiger Erich (2014) Methods Enzymol
Abstract: 
Mitochondrial membrane potential (mtMP) is closely intertwined with oxidative phosphorylation (OXPHOS). The exact nature of the interactions of respiration (flux) and mtMP (force) under various physiological and pathological conditions remains unclear, partially due to methodological limitations. We introduce the combination of high-resolution respirometry and fluorometry with the Oroboros Oxygraph-2k, using the widely applied mtMP indicator safranin. OXPHOS analysis with mouse brain homogenate revealed that safranin inhibits Complex I linked OXPHOS capacity at commonly applied concentrations and targets primarily the phosphorylation system, without effect on LEAK respiration. Complex II linked OXPHOS capacity was inhibited by <20% at 2 µM safranin sufficient for mtMP monitoring. mtMP was higher in the LEAK state without adenylates (LN) than at identical LEAK respiration after ADP stimulation and inhibition by oligomycin (LOmy). Maximum ET capacity was reached in uncoupler titrations before mtMP was fully collapsed, whereas respiration was inhibited at increasing uncoupler concentrations and further reduction of mtMP. Examining a pharmacologically induced state of Complex II dysfunction, mtMP was rather insensitive to 50% inhibition of OXPHOS, but responded strongly to addition of inhibitors when respiration was minimized by substrate depletion. The optimum uncoupler concentration supporting maximum ET capacity varied as a function of pharmacological intervention. Taken together, combined measurement of respiration and mtMP greatly enhances the diagnostic potential of OXPHOS analysis. Respirometric validation of inhibitory and uncoupling effects is mandatory for any fluorophore applied for probing mtMP, in any respiratory state, type of tissue and pathophysiological condition.
• Keywords: High-resolution respirometry, Mitochondrial membrane potential, Safranin, Complex I, Complex II, OXPHOS analysis, electron transfer-pathway
• O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E, AT Innsbruck MitoCom
Labels: MiParea: Respiration, Instruments;methods
Stress:Mitochondrial disease Organism: Mouse Tissue;cell: Nervous system Preparation: Homogenate
Regulation: Uncoupler Coupling state: LEAK, OXPHOS, ET Pathway: N, S, ROX HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol
Safranin, O2k-Demo, O2k-MultiSensor, 1PGM;2D;3U-, SUIT-006, SUIT-020, SUIT-020 Safr mt D036, SUIT-020 Fluo mt D033, SUIT-020 Fluo mt D036, SUIT-006 Fluo mt D034, SUIT-006 O2 mt D022, SUIT-006 O2 D051, SUIT-021, SUIT-021 Fluo mt D036, O2k-brief, BEC 2020.2, MitoFit 2021 MgG, MitoFit 2021 Tissue normoxia, MitoFit 2022 CaG, MitoFit 2022 pmF, MitoFit 2022 NADH, mt-Membrane
SUIT protocols
- O2k-Protocol: »MiPNet20.13 Safranin mt-membranepotential
- Safranin used in the experiments for this publication was Safranin O from Sigma (# S2255, 25 g).
- The fluorescence sensor settings were Amp Polarization voltage = 1000 , gain = 1000 (Figure 9.1) and Amp Polarization voltage = 500 , gain = 1000 (Figures 9.6 and 9.7)
Methods Enzymol
- Galluzzi L, Kroemer G (ed) (2014) Conceptual background and bioenergetic/mitochondrial aspects of oncometabolism. Methods Enzymol 542:509 pp »Bioblast link«
O2k-Publications
O2k-brief
- Bioblast links: Force and membrane potential - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Fundamental relationships
- mt-Membrane potential and protonmotive force
- O2k-Potentiometry
- » O2k-Catalogue: O2k-TPP+ ISE-Module
- » O2k-Manual: MiPNet15.03 O2k-MultiSensor-ISE
- » TPP - O2k-Procedures: Tetraphenylphosphonium
- » Specifications: MiPNet15.08 TPP electrode
- » Poster
- » Unspecific binding of TPP+
- » TPP+ inhibitory effect
- O2k-Potentiometry
- O2k-Fluorometry
- » O2k-Catalogue: O2k-FluoRespirometer
- » O2k-Manual: MiPNet22.11 O2k-FluoRespirometer manual
- » Safranin - O2k-Procedures: MiPNet20.13 Safranin mt-membranepotential / Safranin
- » TMRM - O2k-Procedures: TMRM
- O2k-Fluorometry
- O2k-Publications
Cited by
- 19 articles in PubMed (2021-12-27) https://pubmed.ncbi.nlm.nih.gov/24862266/
- Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. https://doi.org/10.26124/bec:2020-0002
- Cardoso et al (2021) Magnesium Green for fluorometric measurement of ATP production does not interfere with mitochondrial respiration. Bioenerg Commun 2021.1. doi:10.26124/bec:2021-0001
- Komlodi et al (2022) Hydrogen peroxide production, mitochondrial membrane potential and the coenzyme Q redox state measured at tissue normoxia and experimental hyperoxia in heart mitochondria. MitoFit Preprints 2021 (in prep)
- Komlódi et al (2022) The protonmotive force - not merely membrane potential. MitoFit Preprints 2022 (in prep)
