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
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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