MiPNet08.09 CellRespiration

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

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OROBOROS (2016-07-21) Mitochondr Physiol Network

Abstract: Doerrier C, Renner K, Gnaiger E (2003-2016) High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. Mitochondr Physiol Network 08.09(10):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, 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 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: OROBOROS O2k, O2k-Catalogue

O2k-Network Lab: AT_Innsbruck_OROBOROS


Labels: MiParea: Respiration, Instruments;methods Mammal;model: Human Tissue;cell: Blood cells  Preparation: Intact cells Coupling state: LEAK, ROUTINE, ETS HRR: Oxygraph-2k, O2k-Protocol Additional: O2k-Demo, O2k-Core 

» MitoPedia: Respiratory states ROUTINE LEAK ETS ROX

MiPNet0809 FCR.jpg
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).


SUIT protocol

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
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