Cardoso 2022b EBEC

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Luiza Cardoso
Baglivo E, Cardoso LHD, Cecatto C, Gnaiger E (2022) Instrumental quality control and evaluation of mitochondrial respiration medium in high-resolution respirometry with living and permeabilized cells. EBEC 2022.

Link: EBEC 2022 Conference website

Baglivo Eleonora, Cardoso Luiza HD, Cecatto Cristiane, Gnaiger Erich (2022)

Event: EBEC2022 Marseille FR

Quality control is required in research and clinical studies to ensure accuracy and reproducibility. Evaluation of instrumental reproducibility is an essential component of quality control to quantify the precision and limit of detection of an analytical procedure. 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. Instrumental performance of 48 Oroboros O2k-chambers was tested within the 3-year study on MiR05-Kit [1]. Stability of oxygen calibration signals at air saturation and zero oxygen was monitored for up to 8 months. The maximum drift over 1 to 3 days was 0.05 pmol∙s-1∙mL-1. Instrumental O2 background dcO2/dt carried out in the absence of sample was stable within ±1 pmol∙s-1∙mL-1 in the range of experimental O2 concentrations when measured at monthly intervals.

Two substrate-uncoupler-inhibitor titration (SUIT) reference protocols [2] were applied with HEK 293T cells. 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 100 x∙mL-1. The biological variability was higher between cell batches.

MiR05-Kit was stable up to 3 years as shown by consistent cell respiration with five different MiR05 lots. Since many different pathway and coupling control states of living and permeabilized cells were covered in the 2 SUIT protocols [2], 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. The instrumental SOP applied in the present study contributes to the generally applicable internal quality control management ensuring the unique reproducibility in high-resolution respirometry.


Bioblast editor: Plangger M O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E, AT Innsbruck MitoFit


Labels: MiParea: Respiration, Instruments;methods 


Organism: Human  Tissue;cell: HEK  Preparation: Permeabilized cells, Intact cells 



HRR: Oxygraph-2k 


Affiliations

Oroboros Instruments, Innsbruck, Austria. - luiza.cardoso@oroboros.at

References

  1. Baglivo E, Cardoso LHD, Cecatto C, Gnaiger E (2022) Statistical analysis of instrumental reproducibility as internal quality control in high-resolution respirometry. MitoFit Preprints 2022.18. https://doi.org/10.26124/mitofit:2022-0018.v2
  2. Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis, 5th ed. Bioenerg Commun, 2020.2 1−112. https://doi.org/10.26124/bec:2020-0002