Krumschnabel 2016b Abstract MitoFit Science Camp 2016
|O2k-MultiSensor: Mitochondrial respiration media for HRR and simultaneous O2k-Fluorometry.|
Three different mitochondrial respiration media widely differing in chemical composition, MiR05, MiRK03, and Buffer z, were compared regarding their suitability for combined respirometry and fluorometry. Using the Oxygraph-2k in combination with the O2k-Fluo LED2-Module, a substrate-uncoupler-inhibitor-titration (SUIT) protocol was applied to HEK 293T cells and respiration was determined with simultaneous detection of H2O2 production by use of the Amplex Red (AmR) assay system. We observed that respiration was equally well supported by MiR05 and Buffer z, whereas oxygen fluxes detected with MiRK03 were consistently lower at all coupling and substrate states. Estimated H2O2 production rates were largely comparable with all three media, while assay sensitivity was comparatively low but very stable with MiR05, and higher but declining over time in the other two media. A combination of MiR05 and Buffer z, MiR07-T1, proved to support respiration just as well as MiR05, gave similar estimates for H2O2 production rates, and showed AmR assay sensitivity that was comparable to that of MiR05 but slightly less stable over time. Altogether, medium composition appears critical for valid multi-sensor respirometry and fluorometry and requires evaluation for different specific applications.
Labels: MiParea: Respiration, Instruments;methods
Stress:Oxidative stress;RONS Organism: Human Tissue;cell: Kidney, HEK
Coupling state: LEAK, OXPHOS, ET Pathway: N, S, NS, ROX HRR: Oxygraph-2k, O2k-Fluorometer Event: D1 MitoFit Science Camp 2016, Amplex Red
1-OROBOROS INSTRUMENTS, Innsbruck; 2-D. Swarovski Research Lab, Dept Visceral, Transplant and Thoracic Surgery, Innsbruck Med Univ, Austria. - [email protected]