Laner 2015 Abstract MiPschool Cape Town 2015
|The Oroboros Oxygraph-2k for high-resolution respirometry and O2k-Fluorometry: a new quality in OXPHOS analysis.|
Event: MiPschool Cape Town 2015
High-resolution respirometry (HRR) has become established world-wide for the diagnosis of mitochondrial respiratory control and OXPHOS analysis, reflected in >1,400 publications using the Oroboros Oxygraph-2k. The O2k-Fluorometer is a recent extension of the Oxygraph-2k for combining HRR and fluorometric measurements. Multiple titrations are routinely made in substrate-uncoupler-inhibitor titration (SUIT) protocols, which increase the diagnostic information in single assays. Two major applications of O2k-Fluorometry are presented.
H2O2 production measured with Amplex® Ultrared in mouse heart mitochondria and intact yeast cells was not only a function of respiratory substrate and coupling state, but depended strongly on experimental oxygen levels below and even above air saturation of the respiration medium [1,2].
Mitochondrial membrane potential was measured with Safranin (2 µM) with high sensitivity particularly in the range of low membrane potentials. However, Safranin specifically inhibited Complex I linked respiration and the phosphorylation system, such that meaningful applications had to be restricted to Complex II linked respiration with succinate and rotenone. Inhibitory side effects must be critically evaluated for any probe of mitochondrial membrane potential .
These examples illustrate the sensitivity and wide range of applicability of O2k-fluorometry and demonstrate the importance of combining measurements of hydrogen peroxide production and mitochondrial membrane potential with simultaneous monitoring of oxygen concentration and oxygen consumption . The O2k receives the most excellent feedback from its many users, particularly due to its high sensitivity, reliability, and economy with very low running costs.
Labels: MiParea: Instruments;methods, mt-Membrane
Organism: Mouse, Saccharomyces cerevisiae Tissue;cell: Heart Preparation: Intact cells, Isolated mitochondria
Coupling state: LEAK, ROUTINE, OXPHOS, ET Pathway: N, S, NS HRR: Oxygraph-2k, O2k-Fluorometer
1-Oroboros Instruments, Innsbruck
2-D. Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria. - [email protected]
- Fontana-Ayoub M, Fasching M, Eigentler A, Laner V, Gnaiger E (2013) O2k-Fluorometry: Amplex® UltraRed using freeze-dried baker’s yeast. Mitochondr Physiol Network 18.06.
- Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Meth Mol Biol 1264:245-61.
- 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.
- For more details visit: O2k-Fluorometer