Zdrazilova 2022 PLOS ONE

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Zdrazilova L, Hansikova H, Gnaiger E (2022) Comparable respiratory activity in attached and suspended human fibroblasts. PLoS ONE 17:e0264496. https://doi.org/10.1371/journal.pone.0264496

» PMID: 35239701 Open Access

Zdrazilova Lucie, Hansikova Hana, Gnaiger Erich (2022) PLOS ONE

Abstract: 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 versus 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 between attached and suspended cells in ROUTINE respiration of living cells and LEAK respiration obtained after inhibition of ATP synthase by oligomycin. The electron transfer capacity was higher in the O2k than in the XF24. This could be explained by a limitation to two uncoupler titrations in the XF24 which led to an underestimation compared to multiple titration steps in the O2k. A quantitative evaluation of respiration measured via different platforms revealed that short-term suspension of fibroblasts did not affect respiratory activity and coupling control. Evaluation of results obtained by different platforms provides a test for reproducibility beyond repeatability. Repeatability and reproducibility are required for building a validated 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

ORCID: ORCID.png Zdrazilova Lucie, ORCID.png Hanskova Hana, ORCID.png Gnaiger Erich

Data availability

Original files are available Open Access at Zenodo repository: 10.5281/zenodo.5518059

Article processing charge

  • Public Library of Science (PLOS): $ 1749.00

Graphical abstract
"Assuming a publication charge of € 1200 per article, scientists mentioning mitochondria or photosynthesis pay € 100 000 every day in 2021 for ‘selling’ their output to publishers ― over € 36 Mill per year."
- Gnaiger E (2021) Beyond counting papers – a mission and vision for scientific publication. https://doi.org/10.26124/bec:2021-0005
- »Paywall journalism« 


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. https://doi.org/10.1007/978-1-4939-7831-1_32018PMID: 29850993 »O2k-brief
Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. https://doi.org/10.1016/S0034-5687(01)00307-32001Bioblast pdf
Respir Physiol 128:277-97. PMID: 11718759
Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial dysfunction in drug-induced toxicity (Dykens JA, Will Y, eds) John Wiley & Sons, Inc, Hoboken, NJ:327-52.2008Bioblast pdf
O2k-Protocols contents
Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. https://doi.org/10.26124/bec:2020-00022020Open Access pdf published online 2020-12-30

Gnaiger E (2021) Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts. MitoFit Preprints 2021.08. https://doi.org/10.26124/mitofit:2021-00082021MitoFit Preprints 2021.08.
MitoFit pdf
Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts
Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v12020Bioenerg Commun 2020.1. Open Access pdf published online 2020-05-20

Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. https://doi.org/10.1042/BJ200400952004Biochem J 380:919-28. PMID: 15018610 - Open Access
Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-00042021Bioenerg Commun 2021.4. Open Access pdf published online 2021-12-21

Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: facts and artefacts in mitochondrial oxygen kinetics. https://doi.org/10.1007/978-1-4419-1241-1_22010Bioblast pdf
Adv Exp Med Biol 662:7-25. PMID: 20204766 Open Access
Wu M, Neilson A, Swift AL, Moran R, Tamagnine J, Parslow D, Armistead S, Lemire K, Orrell J, Teich J, Chomicz S, Ferrick DA (2007) Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. https://doi.org/10.1152/ajpcell.00247.20062007Am J Physiol Cell Physiol 292:C125-36. PMID: 16971499 Open Access
Ye F, Hoppel CL (2013) Measuring oxidative phosphorylation in human skin fibroblasts. https://doi.org/10.1016/j.ab.2013.02.0102013Anal Biochem 437:52-8. PMID: 23462540
Yépez VA, Kremer LS, Iuso A, Gusic M, Kopajtich R, Koňaříková E, Nadel A, Wachutka L, Prokisch H, Gagneur J (2018) OCR-Stats: Robust estimation and statistical testing of mitochondrial respiration activities using Seahorse XF Analyzer. https://doi.org/10.1371/journal.pone.01999382018PLOS ONE 13:e0199938. PMID: 29995917 Open Access
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O2k Quality Control 1: Polarographic oxygen sensors and accuracy of calibration.
2023-02-06Bioblast pdf  » Versions


Template NextGen-O2k.jpg
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).

MitoFit Preprints

MitoFit Preprints
Comparable respiratory activity in attached and suspended human fibroblasts

MitoFit Preprints 2021.7.

Cited by

Gnaiger E (2021) Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts. MitoFit Preprints 2021.8.

Gnaiger E (2021) Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts. MitoFit Preprints 2021.8. https://doi.org/10.26124/mitofit:2021-0008
  • Krako Jakovljevic N, Ebanks B, Katyal G, Chakrabarti L, Markovic I, Moisoi N (2021) Mitochondrial homeostasis in cellular models of Parkinson’s Disease. Bioenerg Commun 2021.2. https://doi.org/10.26124/bec:2021-0002
  • Komlódi T, Schmitt S, Zdrazilova L, Donnelly C, Zischka H, Gnaiger E. Oxygen dependence of hydrogen peroxide production in isolated mitochondria and permeabilized cells. MitoFit Preprints (in prep).
» Comparison of respirometric methods

On terminology

» Mitochondrial states and rates - terminology beyond MitoEAGLE 2020
For harmonization of terminology on respiratory states and rates, see


MitoEAGLE terminology 

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, Comparison of respirometric methods, MitoFit 2021 BCA, MitoFit 2021 AmR, MitoFit2022QC 

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