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High-resolution respirometry

From Bioblast


high-resolution terminology - matching measurements at high-resolution


High-resolution respirometry

Description

O2k-Fluorometer Series G.jpg

Mitochondrial function and dysfunction have gained an increasing interest over the past ten years, reflecting growing awareness of the fact that mitochondria play a pivotal role in human health and disease. The most suitable way to analyze mitochondrial function is by means of Oxygraph-2k (O2k) high-resolution respirometry applying Substrate-uncoupler-inhibitor titration (SUIT) protocols that allow the diagnosis of numerous mitochondrial pathway defects in a single respirometry assay. Technically, HRR is based on the OROBOROS O2k, combining optimized chamber design, application of oxygen-tight materials, electrochemical sensors, Peltier-temperature control and specially developed software features (DatLab) to obtain the unique sensitive and quantitative resolution of oxygen concentration and oxygen flux, with both, a closed-chamber or open-chamber mode of operation (TIP2k). Standardized calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background oxygen flux (systemic flux compensation) provide the experimental basis for high accuracy of quantitative results and quality control in HRR. The most important extension to the O2k-MultiSensor systems is the O2k-Fluorometer.

Abbreviation: HRR

Reference: Gnaiger 1995 J Bioenerg Biomembr, Gnaiger 2008 POS, Gnaiger 2001 Respir Physiol, Gnaiger 2014 MitoPathways



MitoPedia methods: Respirometry, Fluorometry, Spectrophotometry 


MitoPedia O2k and high-resolution respirometry: DatLab 



Dioxygen


What is an Oxygraph?

  • O2k: The O2k measures oxygen (dioxygen, O2) as a gas dissolved in aqueous solution.
  • With the O2k, experimental data are displayed not only of oxygen concentration over time; but real-time transformation of these data allows the experimenter to follow respiration (oxygen consumption, oxygen flux) of biological and biochemical samples while the experiment is still running.
  • An Oxygraph is not used to measure oxygen concentration in a tissue, but for high-resolution analysis of respiratory activity of biological or biochemical samples transferred into the experimental chamber.


MitoPathways
Pesta and Gnaiger 2012


Specific substrate-uncoupler-inhibitor titration (SUIT) protocols are applied in studies of cell respiration or mitochondrial activity. In experiments with intact cells, different coupling states can be studied to evaluate ROUTINE respiratory activity, respiration coupled to ATP production, respiratory excess capacity, residual oxygen consumption, and, by extension of conventional protocols, intactness of cells. In mitochondrial preparations such as isolated mitochondria, permeabilized cells and tissue samples, or homogenized tissue, quantitative information is obtained on selected mitochondrial pathways is obtained, particularly of the process of oxidative phosphorylation (OXPHOS) in mitochondria and bacteria. Comprehensive OXPHOS analysis leads us beyond the highly informative process of oxygen consumption, when high-resolution respirometry is combined with O2k-MultiSensor modules to simultaneously measure the production of reactive oxygen species or mitochondrial membrane potential, nitric oxide production, acid-base balance or calcium with O2k-Fluorometry or ion selective electrodes. In the O2k, these bioenergetic parameters are always evaluated in combination with the measurement of oxygen concentration and respiration.


O2k-universe.jpg
  • O2k: The O2k is the 2nd generation of our high-resolution respirometers. The first model was available as of 1992. Development of the new O2k started in 2k (2000) and the first O2k specimen was delivered in 2002.


  • O2k: The O2k has two experimental chambers (k: Kammern, German for chambers). Why 2 ml?


  • Power-O2k: Several O2k units are operated simultaneously (P1, P2, ..) to combine high-resolution respirometry with high-output.



MitoPedia: O2k and high-resolution respirometry

» MitoPedia: O2k hardware
» MitoPedia: DatLab
» MitoPedia: SUIT


Links

» MitoPedia: Respiratory states OXPHOS ROUTINE ETS LEAK - ROX
» Respirometry
» MitoPedia: Respirometry
» Wikipedia: Respirometry