Comparison of Mitochondrial Incubation Media for Measurement of Respiration and Hydrogen Peroxide Production

Abstract

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 HO 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. HO 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 HO 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.

Mitochondrial mutations alter endurance exercise response and determinants in mice

“…Mitochondrial respiration and ROS production in soleus muscle were assessed by the Oroboros Oxygraph-2K FluoRespirometer (Oroboros Instruments) as described previously ( 72 ). The Oroboros Oxygraph-2k was first calibrated to air with Mir05Cr respiratory buffer (0.5 mM EGTA, 3 mM MgCl 2 , 60 mM lactobionic acid, 20 mM taurine, 10 mM KH 2 PO 4 , 20 mM Hepes, 110 mM D-sucrose, and fatty acid-free BSA [1 g/L] with the addition of 20 mM creatine and 5 mM DTPA) using the following settings: block temperature, 37 °C; stirrer speed, 750 rpm; oxygen sensor gain, 2; data recording interval, 2 s; gain of fluorescence-module-green, 300.…”

Choice of medium affects PBMC quantification, cell size, and downstream respiratory analysis

“…Optimally, this media should also be used in O2k chambers during HRR analysis. Currently, MiR05 is the preferred medium for HRR analyses as it optimally supports respiration of isolated mitochondrial, tissues and permeabilized cells 17 . However, this media does not prove to be optimal for intact PBMCs based on the significant effects MiR05 has on quantification of the cells shown in this study.…”

Novel mtDNA Imparts the Connective Tissue Disorder of a Tourette Pedigree

“…Respirometry and parallel ROS production using Amplex UltraRed were performed as described previously (26). Briefly, air calibration was performed using the same settings as for intact cells, but in Mir05 respiratory buffer [0.5 mM EGTA, 3 mM MgCl 2 , 60 mM lactobionic acid, 20 mM taurine, 10 mM KH 2 PO 4 , 20 mM HEPES, 110 mM D-sucrose, and fatty acid-free BSA (1 g/l) and 5 mM DTPA].…”