Abstract: Gnaiger E, Renner K (2015) High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. Mitochondr Physiol Network 08.09(09):1-8. »Versions
An experiment on cellular respiration 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 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 ETS 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 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.
• 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
» Instructions for using templates for data evaluation are given in MiPNet08.09 (see above) and MiPNet10.04.