Stability of mitochondrial respiration medium used in high-resolution respirometry with living and permeabilized cells

Quality control (QC) in mitochondrial respiration is essential to ensure accuracy and reproducibility at three levels: (1) sample preparation, (2) instrumental and technical performance, and (3) chemicals and respiration medium. In the present study, we focus on the quality of mitochondrial respiration medium MiR05, which plays a crucial role in supporting mitochondrial function. We prepared MiR05 from 6 lots of MiR05-Kit (Oroboros Instruments) with storage times of 1 to 51 months at room temperature. Two substrate-uncoupler-inhibitor titration (SUIT) reference protocols were used with cryopreserved HEK 293T cells in the Oroboros. These protocols interrogate 20 different respiratory states, of which four are comparable with the following rates: ROUTINE respiration of living cells; residual endogenous respiration after permeabilization by digitonin; electron transfer capacity with succinate, rotenone and glycerophosphate after uncoupler titration; and CIV activity stimulated by ascorbate and TMPD corrected for chemical O2 background. Respiration in all 20 respiratory states was reproducible between the lots of MiR05-Kit and independent of storage time up to 51 months. Chemical O2 background – caused by autoxidation of cytochrome c, ascorbate and TMPD – was a linear function of O2 concentration down to 50 µM and was comparable for each lot of MiR05-Kit and independent of storage time. Our 4-year study demonstrates a constant quality of MiR05-Kit up to several years of storage. Stability of MiR05-Kit and consistency between different lots represent essential elements to obtain reproducible results with high-resolution respirometry in research and clinical investigations.