MiPNet20.14 AmplexRed H2O2-production

O2k-Fluorometry: HRFR and simultaneous determination of H2O2 production with Amplex UltraRed.

»  »Versions

Oroboros (2018-11-26) Mitochondr Physiol Network

Abstract: Krumschnabel G, Fontana-Ayoub M, Fasching M, Gnaiger E (2018) O2k-Fluorometry: HRFR and simultaneous determination of H₂O₂ production with Amplex UltraRed. Mitochondr Physiol Network 20.14(03):1-6.

High-Resolution FluoRespirometry is extended by O2k-MultiSensor modules allowing the simultaneous measurement of oxygen consumption and one additional parameter. 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. Advantages of the simultaneous measurement of additional parameters in a single respirometric chamber:

1. Quality control of preparations by respirometric performance.
2. Objective exclusion criteria for protocol related problems, e.g. overtitration of uncouplers, side effects of TPP⁺ or fluorophores.
3. Elimination of artifacts of normalization for a mitochondrial marker when combining two methods in a single chamber.
4. Additional information is acquired for a limited amount of sample.
5. Oxygen dependence of parameters can be studied.

In the publication by Krumschnabel et al. 2015 we introduce the O2k-Fluo LED2-Module, describe one selected stage in its development, and show its application for the simultaneous measurement of respiration and H₂O₂ production of mouse brain mitochondria. The paper by Makrecka-Kuka et al. 2015 focuses on the application of the Amplex red assay for determining H₂O₂ production in three different models of mitochondrial preparations.

O2k-Publication:

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. Methods Mol Biol 1264:245-61. »Bioblast link«

Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38. »Bioblast link«

• O2k-Network Lab: AT_Innsbruck_Oroboros

Labels: MiParea: Respiration, Instruments;methods 

Organism: Mouse  Tissue;cell: Heart  Preparation: Isolated mitochondria 

Coupling state: LEAK, OXPHOS, ET  Pathway: S  HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol 

O2k-Demo, O2k-MultiSensor, MitoFitPublication