|Zdrazilova L, Hansikova H, Gnaiger E (2021) Comparable respiratory activity in attached and suspended human fibroblasts. MitoFit Preprints 2021.7. doi:10.26124/mitofit:2021-0007|
Abstract: Version 1 (v1) 2021-09-21 doi:10.26124/mitofit:2021-0007
Measurement of oxygen consumption of cultured cells is widely used for diagnosis of mitochondrial diseases, drug testing, biotechnology and toxicology. Fibroblasts are cultured in monolayers but physiological measurements are carried out in suspended or attached cells. We address the question whether respiration differs in attached and suspended cells using multiwell respirometry (Agilent Seahorse XF24) and high-resolution respirometry (Oroboros O2k), respectively. Respiration of human dermal fibroblasts measured in culture medium was baseline-corrected for residual oxygen consumption and expressed as oxygen flow per cell.
No differences were observed in ROUTINE respiration of living cells and LEAK respiration obtained after inhibition of ATP synthase by oligomycin. Multiple steps of uncoupler titrations in the O2k allowed for evaluation of maximum electron transfer capacity, which was higher than respiration obtained in the XF24 due to a limitation to two uncoupler titrations.
Quantitative evaluation of respiration measured in different platforms revealed that short-term suspension of fibroblasts did not affect respiratory activity and coupling control. Consistent results obtained with different platforms provide a test for reproducibility and allow for building an extended respirometric database.
• Keywords: attached cells; ace, suspended cells; sce, fibroblasts, high-resolution respirometry; HRR, Oroboros O2k, Seahorse Bioanalyzer XF24, ROUTINE respiration; R, LEAK respiration; L, electron transfer capacity; E, residual oxygen consumption; Rox, bioenergetic cluster analysis; BCA • Bioblast editor: Gnaiger E • O2k-Network Lab: AT Innsbruck Oroboros, CZ Prague Zeman J
- Original files are available Open Access at Zenodo repository: 10.5281/zenodo.5518059
|Gnaiger 2021 MitoFit BCA||Gnaiger E (2021) Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts. MitoFit Preprints 2021.8. doi:10.26124/mitofit:2021-0008||Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts
- This work was partially funded by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770, NextGen-O2k project, and by Institutional project GAUK110119. Contribution to COST Action CA15203 MitoEAGLE with financial support of Short-Term Scientific missions (LD).
- Gnaiger E (2021) Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts. MitoFit Preprints 2021.8. - »Bioblast link«
Labels: MiParea: Respiration, Instruments;methods
Organism: Human Tissue;cell: Fibroblast Preparation: Intact cells
Regulation: Coupling efficiency;uncoupling Coupling state: LEAK, ROUTINE, ET Pathway: ROX HRR: Oxygraph-2k
SUIT-003, MitoEAGLEPublication, MitoFit 2021 BCA