Laner 2015 Abstract MiPschool Cape Town 2015

From Bioblast
The Oroboros Oxygraph-2k for high-resolution respirometry and O2k-Fluorometry: a new quality in OXPHOS analysis.

Link:

Laner V, Fontana-Ayoub M, Fasching M, Gnaiger E (2015)

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 Oroboros FluoRespirometer is a recent extension of the Oroboros O2k 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 FluoRespirometry are presented.

H2O2 production measured with Amplexยฎ 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 ยตM) 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 [3].

These examples illustrate the sensitivity and wide range of applicability of FluoRespirometry and demonstrate the importance of combining measurements of hydrogen peroxide production and mitochondrial membrane potential with simultaneous monitoring of oxygen concentration and oxygen consumption [4]. The Oroboros receives the most excellent feedback from its many users, particularly due to its high sensitivity, reliability, and economy with very low running costs.


โ€ข O2k-Network Lab: AT Innsbruck Oroboros


Cape Town 2015 Laner Figure.jpg




Affiliations

1-Oroboros Instruments, Innsbruck

2-D. Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria. - verena.laner@oroboros.at

References

  1. Fontana-Ayoub M, Fasching M, Eigentler A, Laner V, Gnaiger E (2013) O2k-Fluorometry: Amplexยฎ UltraRed using freeze-dried bakerโ€™s yeast. Mitochondr Physiol Network 18.06.
  2. 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.
  3. 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.
  4. For more details visit: Oroboros FluoRespirometer


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 



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