Comparison of Mitochondrial Incubation Media for Measurement of Respiration and Hydrogen Peroxide Production
Language English Country United States Media print
Document type Comparative Study, Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't
- Keywords
- Amplex UltraRed, DTPA, HEK 293T cells, High-Resolution FluoRespirometry, Oxygraph-2k, Permeabilized muscle fibers, Respiration media, Substrate-uncoupler-inhibitor titration,
- MeSH
- Cell Respiration MeSH
- Cell Culture Techniques instrumentation methods MeSH
- Fluorescent Dyes chemistry MeSH
- Fluorometry instrumentation methods MeSH
- HEK293 Cells MeSH
- Calibration MeSH
- Culture Media chemistry MeSH
- Humans MeSH
- Mitochondria metabolism MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Oxazines chemistry MeSH
- Cell Membrane Permeability MeSH
- Hydrogen Peroxide metabolism MeSH
- Buffers MeSH
- Sensitivity and Specificity MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
- Names of Substances
- Amplex Red MeSH Browser
- Fluorescent Dyes MeSH
- Culture Media MeSH
- Oxazines MeSH
- Hydrogen Peroxide MeSH
- Buffers MeSH
High-Resolution FluoRespirometry 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.
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