Sobotka 2017 MiPschool Obergurgl

From Bioblast
Ondrej Sobotka
Reference protocols for ROS production measurements.

Link: MitoEAGLE

Sobotka O, Komlodi T, Doerrier C, Gnaiger E (2017)

Event: MiPschool Obergurgl 2017


Substrate uncoupler inhibitor titration (SUIT) protocols allow us to study mitochondrial metabolic pathways and the electron transport system in various physiological and pathological situations. To study mitochondrial respiration, one faces the challenge to obtain valid data for as many respiratory states as possible in one measurement and still have quality control. SUIT protocols give us this opportunity with recently developed Reference protocols 1&2 for a wide use in comparative mitochondrial physiology.

During mitochondrial respiration the oxygen consumption proceeds in cytochrome c oxidase. This simplifies the situation in comparison to ROS production measurements. It is known that the superoxide can be generated on multiple sites over the electron transfer-pathway. Therefore during ROS measurement the question is not only what the rate of this ROS production is, but also its site. Considering this critical issue we face the challenge to specify the exact spot of ROS generation, which is completely different to situation during high-resolution respirometry. To resolve this challenge new approach to respiratory protocol formulation is necessary.

In our presentation we want to review basic concepts, significant milestones and most successful methods in the history of ROS measurement. We want to present our data from simultaneous respiration and hydrogen peroxide assessment using the Amplex Ultra Red assay. Our goal is also to point out and discuss the missteps and pitfalls which could be run into during a measurement by this method. We present our data considering the best possible methodological approach, meaning the optimal media and standard operating procedures.

β€’ Bioblast editor: Kandolf G β€’ O2k-Network Lab: AT Innsbruck Oroboros, CZ Hradec Kralove Cervinkova Z

Labels: MiParea: Respiration 

Enzyme: Complex IV;cytochrome c oxidase 

HRR: Oxygraph-2k, O2k-Protocol  Event: A1, Oral  MitoEAGLE, AmR 

Affiliations and support

Sobotka O(1), Komlodi T(2), Doerrier C(2), Gnaiger E(2)
  1. Dept Physiol, Fac Med Hradec Kralove, Charles Univ, Czech Republic
  2. Oroboros Instruments, Innsbruck, Austria
Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE.
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