High-resolution respirometry: Difference between revisions
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|abbr=HRR | |abbr=HRR | ||
|description=[[Image:O2k-Fluorometer.JPG|180px|left|link=O2k-Fluorometer]] | |description=[[Image:O2k-Fluorometer.JPG|180px|left|link=O2k-Fluorometer]] | ||
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 [[SUIT| substrate-uncoupler-inhibitor titration (SUIT) protocols]] that allow the diagnosis of numerous mitochondrial pathway defects in a single respirometry assay. | 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 [[SUIT| substrate-uncoupler-inhibitor titration (SUIT) protocols]] that allow the diagnosis of numerous mitochondrial pathway defects in a single respirometry assay. Technologically, HRR is based on the [[Oxygraph-2k|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-Module|TIP2k]]). Standardized calibration of the polarographic oxygen sensor ([[MiPNet06.03 POS-calibration-SOP |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]]. | ||
|info=[[Gnaiger 1995 J Bioenerg Biomembr]], [[Gnaiger 2008 POS]], [[Gnaiger 2001 Respir Physiol]], [[Gnaiger 2014 MitoPathways]] | |info=[[Gnaiger 1995 J Bioenerg Biomembr]], [[Gnaiger 2008 POS]], [[Gnaiger 2001 Respir Physiol]], [[Gnaiger 2014 MitoPathways]] | ||
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:::: Specific [[substrate-uncoupler-inhibitor titration]] (SUIT) protocols are applied in studies of cell respiration or mitochondrial activity. In experiments with intact cells, different [[Coupling control state|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 | :::: Specific [[substrate-uncoupler-inhibitor titration]] (SUIT) protocols are applied in studies of cell respiration or mitochondrial activity. In experiments with intact cells, different [[Coupling control state|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 cell membranes. In [[Mitochondrial preparations|mitochondrial preparations]] such as isolated mitochondria, permeabilized cells and tissue samples, or homogenized tissue, quantitative information is obtained on selected [[Gnaiger 2014 MitoPathways |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. | ||
Revision as of 11:32, 20 April 2017
Description
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. Technologically, 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
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.
- 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 cell membranes. 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: 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