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.
» PMID: 24862266 »
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
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
- 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)
- Galluzzi L, Kroemer G (ed) (2014) Conceptual background and bioenergetic/mitochondrial aspects of oncometabolism. Methods Enzymol 542:509 pp »Bioblast link«
- » Keywords: Force and membrane potential