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Difference between revisions of "MiPNet08.09 CellRespiration"

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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, ET-pathway, 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, ET-pathway, ROX.
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/d/da/MiPNet08.09_CellRespiration.pdf |Bioblast pdf]]
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/d/da/MiPNet08.09_CellRespiration.pdf |Bioblast pdf]]  »[http://www.bioblast.at/index.php/File:MiPNet08.09_CellRespiration.pdf Versions]
|authors=Oroboros
|authors=Oroboros
|year=2016-08-11
|year=2016-08-11
|journal=Mitochondr Physiol Network
|journal=Mitochondr Physiol Network
|abstract='''Doerrier C, Gnaiger E (2003-2016) High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ET-pathway, ROX. Mitochondr Physiol Network 08.09(11):1-8.''' »[http://www.bioblast.at/index.php/File:MiPNet08.09_CellRespiration.pdf Versions]
|abstract='''Doerrier C, Gnaiger E (2003-2016) High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ET-pathway, ROX. Mitochondr Physiol Network 08.09(11):1-8.'''


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, ''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-pathway (ET-pathway) at ''J<sub>E</sub>'' of 50 pmol·s­<sup>-1</sup>·ml­<sup>-1</sup>.  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 ET-pathway excess capacity over ROUTINE respiration. The net ROUTINE control ratio, net''R''=(''R-L'')/''E'' was 0.30, indicating that 30% of ET-pathway capacity was activated for ATP production.
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, ''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-pathway (ET-pathway) at ''J<sub>E</sub>'' of 50 pmol·s­<sup>-1</sup>·ml­<sup>-1</sup>.  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 ET-pathway excess capacity over ROUTINE respiration. The net ROUTINE control ratio, net''R''=(''R-L'')/''E'' was 0.30, indicating that 30% of ET-pathway capacity was activated for ATP production.

Revision as of 10:06, 11 January 2018

Publications in the MiPMap
O2k-Protocols contents
High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ET-pathway, ROX.

» Bioblast pdf  »Versions

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, ET-pathway, ROX. Mitochondr Physiol Network 08.09(11):1-8.

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 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-pathway (ET-pathway) at JE of 50 pmol·s-1·ml-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 ET-pathway excess capacity over ROUTINE respiration. The net ROUTINE control ratio, netR=(R-L)/E was 0.30, indicating that 30% of ET-pathway 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, Lymphocyte  Preparation: Intact cells 


Coupling state: LEAK, ROUTINE, ET 

HRR: Oxygraph-2k, O2k-Protocol 

O2k-Demo, O2k-Core, 1ce;2ceOmy;3ceU- 

» MitoPedia: Respiratory states ROUTINE LEAK ET-capacity ROX

MiPNet0809 FCR.jpg

Flux control ratios (FCR) normalized to state ET-pathway (E) in chambers A and B (superimposed). ROUTINE respiration in these cells is regulated at 0.4 of ET-capacity (R/E). Oligomycin (Omy) inhibits respiration to 0.1 ET-capacity (L/E).


Coupling control protocol

Ce1;ce2Omy;ce3U-.png
» Instructions for using templates for data evaluation are given in MiPNet08.09 (see above) and MiPNet10.04 CellRespiration.

ceCCP states

Respiratory state Mark names Explanations
R 1ce ROUTINE
L 2ceOmy Oligomycin
E 3ceU Uncoupler titration
ROX 4ceRotAma 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

Further information

» Intact cells
» Coupling control protocol