MiPNet08.09 CellRespiration

Gnaiger E, Renner K (2003) High-resolution respirometry with cultured cells: A demonstration experiment. Oxygraph-2k workshop report, Schroecken, Austria, March 2003. Mitochondr Physiol Network 08.09.

» Open Access-MiPNet8.09.pdf; Versions

OROBOROS (2003) Mitochondr Physiol Network

Abstract: O2k-Protocol for Intact Cells 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 pmols-1ml-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 pmols1ml-1, while inducing the non-coupled state by FCCP revealed the capacity of the electron transfer system (ETS) at JE of 50 pmols-1ml-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 (respiratory control ratio, RCR=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.

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).

>> O2k-Protocols:Overall contents

>> 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 

Further information

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

>> Excel Templates and DatLab-Demo Files

• Version 7: 2011-12-11
• Version 1: 2003