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High-Resolution Respirometry in a Small-Volume Chamber

Leo, Elettra ; Rychtarova, Lucie ; Garcia-Souza, Luiz F. LU ; Åsander Frostner, Eleonor LU orcid ; Elmér, Eskil LU orcid and Gnaiger, Erich (2025) In Journal of Visualized Experiments 2025(221).
Abstract

Investigating respiratory fluxes is decisive for understanding the complex interplay between metabolic processes. Studies of cells and tissues with limited sample availability and low respiratory rates may benefit from small experimental volumes. We examined if the 0.5-mL chamber yields results consistent with the 2.0-mL chamber at identical sample concentrations used in high-resolution respirometry with the Oroboros. The background O2 flux was 4-fold higher in 0.5-mL than 2.0-mL chambers at air saturation but was reproducible, allowing for accurate background correction. The optimal stirring speed was between 550 rotations per minute (rpm) and 750 rpm in the 0.5-mL chamber. Respiratory fluxes in living cells, permeabilized... (More)

Investigating respiratory fluxes is decisive for understanding the complex interplay between metabolic processes. Studies of cells and tissues with limited sample availability and low respiratory rates may benefit from small experimental volumes. We examined if the 0.5-mL chamber yields results consistent with the 2.0-mL chamber at identical sample concentrations used in high-resolution respirometry with the Oroboros. The background O2 flux was 4-fold higher in 0.5-mL than 2.0-mL chambers at air saturation but was reproducible, allowing for accurate background correction. The optimal stirring speed was between 550 rotations per minute (rpm) and 750 rpm in the 0.5-mL chamber. Respiratory fluxes in living cells, permeabilized cells, and isolated mitochondria were measured in parallel in the 0.5-mL and 2.0-mL chambers, using Substrate-Uncoupler-Inhibitor Titration protocols with 14 to 20 titrations. O2 fluxes in the different chamber types were identical within the limits of detection. The 0.5-mL chamber, requiring close to four times less sample than the 2.0-mL chamber, offers a significant advantage for studies with limited amounts of sample or low respiratory capacities.

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author
; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Visualized Experiments
volume
2025
issue
221
article number
e67442
publisher
MyJoVE Corporation
external identifiers
  • scopus:105013347461
  • pmid:40788866
ISSN
1940-087X
DOI
10.3791/67442
language
English
LU publication?
yes
id
050f822a-f3c6-40cb-8c10-9850c9053c68
date added to LUP
2025-11-11 10:03:08
date last changed
2025-11-12 03:17:19
@article{050f822a-f3c6-40cb-8c10-9850c9053c68,
  abstract     = {{<p>Investigating respiratory fluxes is decisive for understanding the complex interplay between metabolic processes. Studies of cells and tissues with limited sample availability and low respiratory rates may benefit from small experimental volumes. We examined if the 0.5-mL chamber yields results consistent with the 2.0-mL chamber at identical sample concentrations used in high-resolution respirometry with the Oroboros. The background O<sub>2</sub> flux was 4-fold higher in 0.5-mL than 2.0-mL chambers at air saturation but was reproducible, allowing for accurate background correction. The optimal stirring speed was between 550 rotations per minute (rpm) and 750 rpm in the 0.5-mL chamber. Respiratory fluxes in living cells, permeabilized cells, and isolated mitochondria were measured in parallel in the 0.5-mL and 2.0-mL chambers, using Substrate-Uncoupler-Inhibitor Titration protocols with 14 to 20 titrations. O<sub>2</sub> fluxes in the different chamber types were identical within the limits of detection. The 0.5-mL chamber, requiring close to four times less sample than the 2.0-mL chamber, offers a significant advantage for studies with limited amounts of sample or low respiratory capacities.</p>}},
  author       = {{Leo, Elettra and Rychtarova, Lucie and Garcia-Souza, Luiz F. and Åsander Frostner, Eleonor and Elmér, Eskil and Gnaiger, Erich}},
  issn         = {{1940-087X}},
  language     = {{eng}},
  number       = {{221}},
  publisher    = {{MyJoVE Corporation}},
  series       = {{Journal of Visualized Experiments}},
  title        = {{High-Resolution Respirometry in a Small-Volume Chamber}},
  url          = {{http://dx.doi.org/10.3791/67442}},
  doi          = {{10.3791/67442}},
  volume       = {{2025}},
  year         = {{2025}},
}