MiPNet08.09 CellRespiration: Difference between revisions
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{{Publication | {{Publication | ||
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]] High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. | |title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]] High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. [[Media:MiPNet08.09 CellRespiration.pdf|»Bioblast pdf«]] | ||
|info=[http://www.bioblast.at/index.php/File:MiPNet08.09_CellRespiration.pdf Versions] | |||
|authors=OROBOROS | |authors=OROBOROS | ||
|year=2015-01-11 | |year=2015-01-11 | ||
|journal=Mitochondr Physiol Network | |journal=Mitochondr Physiol Network | ||
|abstract= | |abstract=Gnaiger E, Renner K (2015) High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. Mitochondr Physiol Network 08.09(09):1-8. | ||
An experiment on cellular respiration is reported from an O2k workshop on high-resolution respirometry. Leukemia cells were incubated at a density of 1 million cells/ml in 2 ml culture medium in two O2k-Chambers operated in parallel. Cellular ROUTINE respiration, ''J''<sub>R</sub>, resulted in volume-specific oxygen consumption of 20 pmol·s<sup>-1</sup>·ml<sup>-1</sup>. Oxygen concentration changed by merely 6.4 and 6.5 µM in the two O2k-Chambers over a period of 5 min (<1% air saturation per minute). Inhibition by oligomycin (''J<sub>L</sub>''), and rotenone (residual oxygen consumption, ''J''<sub>ROX</sub>; after uncoupling) reduced respiration to 5 and 1 pmol·s<sup>-1</sup>·ml<sup>-1</sup>, while inducing the noncoupled state by the uncoupler FCCP revealed the capacity of the electron transfer system (ETS) at ''J<sub>E</sub>'' of 50 pmol·s<sup>-1</sup>·ml<sup>-1</sup>. The ROUTINE flux control ratio, ''R/E'', was 0.39 (uncoupling control ratio, UCR=''E/R''=2.6), and the LEAK flux control ratio, ''L/E'', was 0.09 (''E/L''=12.0). This indicates tight coupling of OXPHOS, and a large ETS excess capacity over ROUTINE respiration. The net ROUTINE flux control ratio, net''R''=(''R-L'')/''E'' was 0.30, indicating that 30% of ETS capacity was activated for ATP production. | An experiment on cellular respiration is reported from an O2k workshop on high-resolution respirometry. Leukemia cells were incubated at a density of 1 million cells/ml in 2 ml culture medium in two O2k-Chambers operated in parallel. Cellular ROUTINE respiration, ''J''<sub>R</sub>, resulted in volume-specific oxygen consumption of 20 pmol·s<sup>-1</sup>·ml<sup>-1</sup>. Oxygen concentration changed by merely 6.4 and 6.5 µM in the two O2k-Chambers over a period of 5 min (<1% air saturation per minute). Inhibition by oligomycin (''J<sub>L</sub>''), and rotenone (residual oxygen consumption, ''J''<sub>ROX</sub>; after uncoupling) reduced respiration to 5 and 1 pmol·s<sup>-1</sup>·ml<sup>-1</sup>, while inducing the noncoupled state by the uncoupler FCCP revealed the capacity of the electron transfer system (ETS) at ''J<sub>E</sub>'' of 50 pmol·s<sup>-1</sup>·ml<sup>-1</sup>. The ROUTINE flux control ratio, ''R/E'', was 0.39 (uncoupling control ratio, UCR=''E/R''=2.6), and the LEAK flux control ratio, ''L/E'', was 0.09 (''E/L''=12.0). This indicates tight coupling of OXPHOS, and a large ETS excess capacity over ROUTINE respiration. The net ROUTINE flux control ratio, net''R''=(''R-L'')/''E'' was 0.30, indicating that 30% of ETS capacity was activated for ATP production. |
Revision as of 14:44, 18 May 2015
High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. »Bioblast pdf« |
» Versions
OROBOROS (2015-01-11) Mitochondr Physiol Network
Abstract: Gnaiger E, Renner K (2015) High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. Mitochondr Physiol Network 08.09(09):1-8.
An experiment on cellular respiration is reported from an O2k workshop on high-resolution respirometry. Leukemia cells were incubated at a density of 1 million cells/ml in 2 ml culture medium in two O2k-Chambers operated in parallel. Cellular ROUTINE respiration, JR, resulted in volume-specific oxygen consumption of 20 pmol·s-1·ml-1. Oxygen concentration changed by merely 6.4 and 6.5 µM in the two O2k-Chambers over a period of 5 min (<1% air saturation per minute). Inhibition by oligomycin (JL), and rotenone (residual oxygen consumption, JROX; after uncoupling) reduced respiration to 5 and 1 pmol·s-1·ml-1, while inducing the noncoupled state by the uncoupler FCCP revealed the capacity of the electron transfer system (ETS) at JE of 50 pmol·s-1·ml-1. The ROUTINE flux control ratio, R/E, was 0.39 (uncoupling control ratio, UCR=E/R=2.6), and the LEAK flux control ratio, L/E, was 0.09 (E/L=12.0). This indicates tight coupling of OXPHOS, and a large ETS excess capacity over ROUTINE respiration. The net ROUTINE flux control ratio, netR=(R-L)/E was 0.30, indicating that 30% of ETS capacity was activated for ATP production.
Automatic correction for instrumental background amounted to 13% for ROUTINE respiration, but to >50% and 180% for JL and JROX, respectively, illustrating the importance of real-time correction. The experiment illustrates the sensitivity and resproducibility of high-resolution respirometry with the OROBOROS O2k. Calibrations and routine corrections provide the basis of the high accuracy required for mitochondrial respiratory physiology. Real-time analyses were performed, combining high-resolution with instant diagnostic information. In this update graphs are presented illustrating some features of DatLab.
- >> Product: OROBOROS O2k, O2k-Catalogue
• O2k-Network Lab: AT_Innsbruck_OROBOROS
Labels: MiParea: Respiration, Instruments;methods
Organism: Human
Tissue;cell: Blood cells
Preparation: Intact cells
Coupling state: LEAK, ROUTINE, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.
HRR: Oxygraph-2k, Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.
O2k-Demo, O2k-Core
» MitoPedia: Respiratory states
Further information
» Instructions for using templates for data evaluation are given in MiPNet08.09 (see above) and MiPNet10.04.