MiPNet08.09 CellRespiration

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

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OROBOROS (2014-03) Mitochondr Physiol Network

Abstract: Gnaiger E, Renner K (2014) High-resolution respirometry and phosphorylation control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. Mitochondr Physiol Network 08.09(08): 1-8.

An experiment on cellular respiration is reported from an Oxygraph-2k 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 non-coupled state by FCCP revealed the capacity of the electron transfer system (ETS) at JE of 50 pmol*s^-1*ml^-1. The ROUTINE flux control ratio, R/E, was 0.39 (uncoupling control ratio, UCR=E/R=2.6), and the LEAK flux control ratio, L/E, was 0.09 (E/L=12.0). This indicates tight coupling of OXPHOS, and a large respiratory excess capacity over ROUTINE respiration. The net ROUTINE flux 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 Oxygraph-2k. 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 Oxygraph-2k, 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, Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property. 

O2k-Demo, O2k-Core 

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 0.39 of ETS capacity (R/E). Oligomycin (Omy) inhibits respiration to 0.08 ETS capacity (L/E).

Further information

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

>> Excel Templates and DatLab-Demo Files