Komlodi 2018 Methods Mol Biol
|Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55.|
Abstract: High-resolution respirometry is a well-established and versatile approach to study mitochondrial oxygen uptake amperometrically in combination with measurement of fluorescence signals. One of the most frequently applied fluorescent dyes is Amplex UltraRed for monitoring rates of hydrogen peroxide production. Selection of an appropriate mitochondrial respiration medium is of crucial importance, the primary role of which is to support and preserve optimum mitochondrial function. For harmonization of results in a common database, we compared respiration and H2O2 production of permeabilized HEK 293T cells measured in MiR05 (sucrose and K-lactobionate), Buffer Z (K-MES and KCl), MiR07 (combination of MiR05 and Buffer Z), and MiRK03 (KCl). Respiration in a simple substrate-uncoupler-inhibitor titration protocol was identical in MiR05, Buffer Z, and MiR07, whereas oxygen fluxes detected with MiRK03 were consistently lower in all coupling and electron transfer-pathway states. H2O2 production rates were comparable in all four media, while assay sensitivity was comparatively low with MiR05 and MiR07 and higher but declining over time in the other two media. Stability of assay sensitivity over experimental time was highest in MiR05 but slightly less in MiR07. Taken together, MiR05 and Buffer Z yield comparable results on respiration and H2O2 production. Despite the lower sensitivity, MiR05 was selected as the medium of choice for FluoRespirometry due to the highest stability of the sensitivity or calibration constant observed in experiments over periods of up to 2 h.
• Keywords: Amplex UltraRed, high-resolution respirometry, Oxygraph-2k, Respiration media, Substrate-uncoupler-inhibitor titration, HEK 293T cells, Permeabilized muscle fibers, DTPA, Buffer z • Bioblast editor: Krumschnabel G • O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck Oroboros
Labels: MiParea: Respiration, Instruments;methods
Organism: Human, Mouse Tissue;cell: Skeletal muscle, HEK Preparation: Permeabilized cells, Permeabilized tissue
Coupling state: LEAK, OXPHOS, ET Pathway: F, N, S, CIV, NS, ROX HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol
AmR, 2018-07, MitoEAGLEPublication, SUIT-009 AmR mt D021, SUIT-013, SUIT-013 AmR ce D023, SUIT-009 AmR pce D019, SUIT-018, SUIT-018 AmR mt D031, SUIT-006 AmR mt D048, SUIT-003 AmR ce D017, SUIT-003 AmR ce D058, MitoFit 2021 AmR, MitoFit 2021 Dark respiration, MitoFit 2021 Photosynthesis, MitoFit 2021 Yeast
- Sobotka et al (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed in yeast cells. MitoFit Preprints 2021.
- Komlodi T et al (2021) Oxygen dependence of hydrogen peroxide production using Amplex UltraRed in yeast cells, isolated mitochondria, and permeabilized cells. MitoFit Preprints 2021 (in prep).
- Huete-Ortega et al (2021) Substrate-uncoupler-inhibitor-titration protocols for dark respiration in Chlamydomonas reinhardtii. MitoFit Preprints 2021 (in prep).
- Huete-Ortega et al (2021) Substrate-uncoupler-inhibitor-titration protocols for photosynthesis in Chlamydomonas reinhardtii. MitoFit Preprints 2021 (in prep).