MiPNet08.09 CellRespiration: Difference between revisions
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== Coupling control protocol == | == Coupling control protocol == | ||
[[File:1R,2Omy,3U-.jpg|right|300px|link= | [[File:1R,2Omy,3U-.jpg|right|300px|link=1ce;2Omy;3U-]] | ||
****: CCP: RLE [[ | ****: CCP: RLE [[1ce;2Omy;3U-]] | ||
::::ยป Instructions for using templates for data evaluation are given in MiPNet08.09 (see above) and [[MiPNet10.04 CellRespiration]]. | ::::ยป Instructions for using templates for data evaluation are given in MiPNet08.09 (see above) and [[MiPNet10.04 CellRespiration]]. | ||
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Revision as of 06:52, 26 June 2017
High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. |
OROBOROS (2016-08-11) Mitochondr Physiol Network
Abstract: Doerrier C, Gnaiger E (2003-2016) High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. Mitochondr Physiol Network 08.09(11):1-8. ยปVersions
An experiment on respiration of intact cells 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 pmolยทs-1ยทml-1. Oxygen concentration changed by merely 6.4 and 6.5 ยตM 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 pmolยทs-1ยทml-1, while inducing the noncoupled state by the uncoupler FCCP revealed the capacity of the electron transfer system (ETS) at JE of 50 pmolยทs-1ยทml-1. The ROUTINE control ratio, R/E, was 0.4 (uncoupling control ratio, UCR=E/R=2.5), and the LEAK control ratio, L/E, was 0.1 (E/L=12.0). This indicates tight coupling of OXPHOS, and a large ETS excess capacity over ROUTINE respiration. The net ROUTINE 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: Oxygraph-2k, O2k-Catalogue
โข O2k-Network Lab: AT_Innsbruck_OROBOROS
Labels: MiParea: Respiration, Instruments;methods
Organism: Human
Tissue;cell: Blood cells, Lymphocyte
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, O2k-Protocol
O2k-Demo, O2k-Core, CCP02
ยป MitoPedia: Respiratory states
Flux control ratios (FCR) normalized to state ETS (E) in chambers A and B (superimposed). ROUTINE respiration in these cells is regulated at 0.4 of ETS capacity (R/E). Oligomycin (Omy) inhibits respiration to 0.1 ETS capacity (L/E).
Coupling control protocol
- CCP: RLE 1ce;2Omy;3U-
- ยป Instructions for using templates for data evaluation are given in MiPNet08.09 (see above) and MiPNet10.04 CellRespiration.
CCP states
Respiratory state | Mark names | Explanations |
---|---|---|
R | 1ce | ROUTINE |
L | 2Omy | Oligomycin |
E | 3U | Uncoupler titration |
ROX | 4Ama | Rotenone, Antimycin A, residual oxygen consumption |
DatLab-Analysis templates and DatLab Demo Files
DatLab 7
- Calibration-File: MiPNet08.09_CellRespiration_Calib.DLD
- Demo-File: MiPNet08.09_CellRespiration.DLD
- DatLab-Analysis template: CCP02.xlsx
DatLab 6
- Calibration-File: MIPNET08.09_2003-03-29_P1-01_CALIB.DLD
- Demo-File: MIPNET08.09_2003-03-29_P1-02_CELLS.DLD
- Background Excel demo and template: MiPNet08.09_O2k-Background_Cells.xlsx
- DatLab-Analysis template: MiPNet08.09_O2k-Analysis_Cells.xlsx
DatLab 5
- Calibration-File: MIPNET08.09_2003-03-29_P1-01_CALIB.DLD
- Demo-File: MIPNET08.09_2003-03-29_P1-02_CELLS.DLD
- Background Excel demo and template: O2k-Background_Cells_0809.xlsx
- DatLab-Analysis template: O2k-Analysis_Cells_0809.xlsx