Baglivo 2022a ESCI Bari
Event: ESCI 2022 Bari IT
Quality control is required in clinical studies to ensure accuracy and reproducibility. The technical repeatability of subsamples reflects instrumental resolution and experimental precision, but is insufficiently reported in mitochondrial respiratory research. A frequently neglected problem is quality control of chemicals and incubation media. Mitochondrial respiration medium MiR05 is prepared from MiR05-Kit (Oroboros Instruments), which contains 7 chemicals and is stored as a crystalline powder. The powder is dissolved in H2O, bovine serum albumin (BSA) is added, and the pH is adjusted.
We evaluated the quality of 5 lots of MiR05-Kit stored at room temperature for 2 months to 3 years. Two substrate-uncoupler-inhibitor titration (SUIT) reference protocols  were applied in the Oroboros O2k with cryopreserved HEK293T cells. Compared to the instrumental limit of detection of volume-specific O2 flux of ±1 pmol·s-1·mL-1, the range of technical repeats in ROUTINE respiration was ±5 pmol·s-1·mL-1 or ±15 % using 8-16 O2k chambers in parallel at a cell concentration of 106 x∙mL-1. The biological variability between cell batches was higher. Homogenous stocks of suspended cells were used to compare MiR05 lots at 3 annual intervals.
The MiR05-Kit was stable up to 3 years as shown by consistent cell respiration with different MiR05 lots. Since many different pathway and coupling control states of living and permeabilized cells were covered in the 2 SUIT protocols , these results exclude specific inhibitory modifications of MiR05 during storage.
Our study demonstrates a constant quality of MiR05-Kit up to 3 years of storage. This represents an important quality control in high-resolution respirometry to obtain reliable and reproducible results in scientific and clinical investigations.
• Keywords: Mitochondrial respiration medium, Cryopreserved HEK cells, Living and permeabilized cells, Quality control, Reproducibility • Bioblast editor: Plangger M • O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E, AT Innsbruck MitoFit
- Oroboros Instruments GmbH, Oroboros Ecosystem, Innsbruck, Austria.
- Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70.
List of abbreviations, terms and definitions - MitoPedia
Labels: MiParea: Respiration, Instruments;methods
Organism: Human Tissue;cell: HEK Preparation: Permeabilized cells, Intact cells