Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

MiPNet08.09 CellRespiration

From Bioblast
(Redirected from MiPNet08.09)
Publications in the MiPMap
O2k-Procedures contents
High-resolution respirometry and coupling-control protocol with living cells: ROUTINE, LEAK, ET-pathway, ROX.

Bioblast pdf  禄Versions

Oroboros (2016-08-11) Mitochondr Physiol Network

Abstract: Doerrier C, Gnaiger E (2003-2016) High-resolution respirometry and coupling-control protocol with living cells: ROUTINE, LEAK, ET-pathway, ROX. Mitochondr Physiol Network 08.09(11):1-8.

An experiment on respiration of living 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路s-1路ml-1. Oxygen concentration changed by merely 6.4 and 6.5 碌M 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路s-1路ml-1, while inducing the noncoupled state by the uncoupler FCCP revealed the capacity of the Electron transfer-pathway (ET-pathway) at JE of 50 pmol路s-1路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 ET-pathway excess capacity over ROUTINE respiration. The net ROUTINE control ratio, netR=(R-L)/E was 0.30, indicating that 30% of ET-pathway 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.

禄 Product: Oroboros O2k, O2k-Catalogue

Bioblast editor: Gnaiger E O2k-Network Lab: AT_Innsbruck_Oroboros


Labels: MiParea: Respiration, Instruments;methods 


Organism: Human  Tissue;cell: Blood cells, Lymphocyte  Preparation: Intact cells 


Coupling state: LEAK, ROUTINE, ET 

HRR: Oxygraph-2k, O2k-Protocol 

O2k-Demo, O2k-Core, SUIT-003 O2 ce D009, SUIT-003, SUIT-006 General O2 pce D053, Leukemia 

MitoPedia: Respiratory states ROUTINE LEAK ET capacity ROX

MiPNet0809 FCR.jpg

Flux control ratios (FCR) normalized to state ET-pathway (E) in chambers A and B (superimposed). ROUTINE respiration in these cells is regulated at 0.4 of ET capacity (R/E). Oligomycin (Omy) inhibits respiration to 0.1 ET capacity (L/E).


Coupling-control protocol

Ce1;ce2Omy;ce3U-.png
禄 Instructions for using templates for data evaluation are given in MiPNet08.09 (see above) and MiPNet10.04 CellRespiration.

ceCCP states

Respiratory state Mark names Explanations
R 1ce ROUTINE
L 2ceOmy Oligomycin
E 3ceU Uncoupler titration
ROX 4ceRotAma Rotenone, Antimycin A, residual oxygen consumption






DatLab-Analysis templates and DatLab Demo Files

DatLab 7

Calibration-File: MiPNet08.09_CellRespiration_Calib.DLD
Demo-File: MiPNet08.09_CellRespiration.DLD
DatLab-Analysis template: CCP02.xlsx

DatLab 6

Calibration-File: MIPNET08.09_2003-03-29_P1-01_CALIB.DLD
Demo-File: MIPNET08.09_2003-03-29_P1-02_CELLS.DLD
Background Excel demo and template: MiPNet08.09_O2k-Background_Cells.xlsx
DatLab-Analysis template: MiPNet08.09_O2k-Analysis_Cells.xlsx

DatLab 5

Calibration-File: MIPNET08.09_2003-03-29_P1-01_CALIB.DLD
Demo-File: MIPNET08.09_2003-03-29_P1-02_CELLS.DLD
Background Excel demo and template: O2k-Background_Cells_0809.xlsx
DatLab-Analysis template: O2k-Analysis_Cells_0809.xlsx

Further information

Living cells
Coupling-control protocol