Comparable respiratory activity in attached and suspended human fibroblasts
Open accessAbstract
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.
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Respiratory rates measured at basal condition were similar between suspended and attached astrocytes.
“Of note, the respiratory rates measured at basal condition were similar between suspended and attached astrocytes (Figure 2B,D vs. Figure 3A,B), in line with previous observations on human fibroblasts [46].”
From Proliferating Astrocytes in Primary Culture Do Not Depend upon Mitochondrial Respiratory Complex I Activity or Oxidative Phosphorylation (2023) · cites this paper as [46]
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Cited by
Sorted by citations
Silva, E. A., Dalla Costa, A. P., Ruas, J. S., Siqueira-Santos, E. S., Francisco, A., & Castilho, R. F. (2023) · Cells · doi:10.3390/cells12050683 · Cited by 5 · see citing works
- Authors
- Zdrazilova, L., Hansikova, H., & Gnaiger, E.
- Affiliations
- Oroboros Instruments (Austria) (AT)
- Corresponding author
- Gnaiger, Erich
- Venue
- PLOS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2022-03-03
- Volume
- 17
- Issue
- 3
- Pages
- e0264496
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC8893708
- OpenAlex
- W4214825164
- Cited by
- 28
- References
- 3
- Citations by year
- 2026: 5 · 2025: 6 · 2024: 8 · 2023: 7 · 2022: 2
- FWCI
- 1.88
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- PLoS ONE (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- European Union’s Horizon 2020 research and innovation program (859770, NextGen-O2k project)
- Institutional project of Charles University (GAUK110119, SVV–UK 260367)
- Ministry of Health of the Czech Republic (NV19-07-00149)
- COST Action (CA15203 MitoEAGLE)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
- Physical Sciences › Chemistry › Physical and Theoretical Chemistry › thermodynamics and calorimetric analyses
- Keywords
- cellular respiration, human dermal fibroblasts, high-resolution respirometry, mitochondrial function, electron transfer capacity, oligomycin, ATP synthase inhibition, reproducibility
- MeSH terms
- Fibroblasts, Humans, Oxidative Phosphorylation, Oxygen Consumption, Reproducibility of Results, Cell Respiration
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