MiPNet08.09 CellRespiration

High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ETS, ROX.

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OROBOROS (2016-07-20) Mitochondr Physiol Network

Abstract: Doerrier C, Renner K, Gnaiger E (2003-16) High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. Mitochondr Physiol Network 08.09(10 in prep):1-8. »Versions

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_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 system (ETS) 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 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 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

• 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, O2k-Protocol 

O2k-Demo, O2k-Core 

» MitoPedia: Respiratory states

The figure represents flux control ratios (FCR) normalized to state ETS (E) in chambers A and B. ROUTINE respiration in these cells is regulated at 0.4 of ETS capacity (R/E). Oligomycin (Omy) inhibits respiration to 0.9 ETS capacity.

SUIT protocol

File:Ce R 1Omy 2U 3RotAma.png

» SUIT protocol: SUIT MiPNet08.09 CellRespiration

» Instructions for using templates for data evaluation are given in MiPNet08.09 (see above) and MiPNet10.04 CellRespiration.

SUIT states

Respiratory state	Coupling control
R	ROUTINE
1Omy	Omy(L)
2U	Uncoupler(E)
3Ama	Antimycin A(ROX)

Further information

» Intact cells

» Excel Templates and DatLab-Demo Files

» Coupling control protocol