MiPNet08.09 CellRespiration

High-resolution respirometry and coupling-control protocol with living cells: ROUTINE, LEAK, ET-pathway, ROX.

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Oroboros (2016-08-11) Mitochondr Physiol Network

Abstract: Doerrier C, Gnaiger E (2003-2016) High-resolution respirometry and coupling-control protocol with living cells: ROUTINE, LEAK, ET-pathway, ROX. Mitochondr Physiol Network 08.09(11):1-8.

An experiment on respiration of living 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_R, 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 (J_L), and rotenone (residual oxygen consumption, J_ROX; 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 J_E 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 J_L and J_ROX, 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

• Bioblast editor: Gnaiger E • 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, SUIT-003 O2 ce D009, SUIT-003, SUIT-006 General O2 pce D053, Leukemia 

» MitoPedia: Respiratory states

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

• • • • ceCCP: RLE 1ce;2ceOmy;3ceU-

» 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

» Living cells

» Coupling-control protocol