Fasching 2011 Abstract Berlin

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Fasching M, Harrison DK, Tretter L, Gnaiger E (2011) Combination of high-resolution respirometry and fluorometry for continuous monitoring of hydrogen peroxide production by mitochondria with resolution in the nanomolar range. Abstract Berlin.

Link: MiP-Poster, MiP2011 Bordeaux, FR

Fasching M, Harrison DK, Tretter L, Gnaiger E (2011)

Event: Targeting Mitochondria Berlin

Combining fluorometric methods with high-resolution respirometry enabled a wide range of analytical parameters of major interest in mitochochondrial physiology. The advantages of measuring two parameters simultaneously are discussed and we describe the simultaneous high-resolution measurement of oxygen consumption and fluorometric determination of hydrogen peroxide production in the O2k, using Amplex Red®.

MiPNetLab: AT Innsbruck Gnaiger E, HU Budapest Tretter L


Labels: MiParea: Respiration, Instruments;methods Mammal;model: Mouse Tissue;cell: Nervous system  Preparation: Isolated mitochondria HRR: Oxygraph-2k, O2k-Fluorometer 

Full abstract

High-resolution respiromety [1] is extended by O2k-MultiSensor techniques allowing the simultaneous measurement of oxygen consumption and one additional parameter (mitochondrial membrane potential, pH, Ca2+, or NO concentration [2,3]). Combining optical measurements with high-resolution respirometry further increased the range of analytical opportunities. In particular, fluorometric methods are available for a wide range of analytical parameters of major interest in mitochochondrial physiology: H2O2 production, mitochondrial membrane potential, intracellular pH, Ca2+, Mg2+, and NADH levels.

The simultaneous measurement of additional parameters in a single respirometric chamber under strictly identical conditions offers important advantages: (i) respirometric performance provides a quality control of cells or mitochondrial preparations; (ii) overtitration of uncouplers, incomplete action of inhibitors, or non-saturating substrate concentrations are evaluated by the simultaneous response of multiple parameters, providing objective exclusion criteria; (iii) side effects of TPP+ or fluorophores (inhibition, dyscoupling) are detected by respiration and artifacts are, therefore, excluded; (iv) the direct relationship between the different parameters eliminates artifacts of normalization for a mitochondrial marker; (v) additional information is acquired for a limited amount of sample. In addition, there are practical and economical advantages, saving handling time and money.

The glass chamber of the OROBOROS Oxygraph-2k (O2k) allows transmitting optical signals through the chamber wall. This extends MultiSensor applications, obtaining the optical signal in addition to the signals of the oxygen sensor and of other electrodes inserted through the stopper (e.g. respiration, mitochondrial membrane potential and H2O2 production).

We describe the simultaneous high-resolution measurement of oxygen consumption and fluorometric determination of hydrogen peroxide production in the O2k, using Amplex Red®. Sensitivity to detect H2O2 production in relation to signal drift and noise is optimized. Low nanomolar concentrations of hydrogen peroxide were detected by integrating the components of a fluorometer into the O2k. Potential problems are discussed for a three-parameter measurement, e.g. interaction of fluorophores with the TPP+ electrode.

Supported by MitoCom Tyrol.


[1] Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds.) John Wiley:327-52.

[2] Aguirre E, Rodríguez-Juárez F, Bellelli A, Gnaiger E, Cadenas S (2010) Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells. Biochim. Biophys. Acta 1797:557-65.

[3] http://www.oroboros.at/?O2k-MultiSensor

[4] Original presentation at MiP2011

Erratum

In the description of the media the concentration of HEPES is stated as "20 M". This should read "20 mM". --Fasching Mario 09:26, 29 May 2012 (CEST)

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