MiPNet10.04 CellRespiration

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An experiment with high-resolution respirometry: coupling control in cell respiration.

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Oroboros (2016-08-08) Mitochondr Physiol Network

Abstract: Doerrier C, Gnaiger E (2005-2016) An experiment with high-resolution respirometry: Coupling control in cell respiration. Mitochondr Physiol Network 10.04(09):1-12.

Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with cultured, suspended cells in the Oroboros O2k. The experiment demonstrates manual titrations of inhibitors, and automatic titrations of an uncoupler using the electronic Titration-Injection microPump TIP2k. Application of the DatLab 7 software is shown for instrumental control (O2k and TIP2k), and real-time data analysis. The following guideline describes the experiment in the form of a laboratory protocol, complementary to the relevant sections of the O2k Manual. The experiments were carried out by participants of an O2k-Workshop on high-resolution respirometry in April 2005 (IOC30; Schröcken, Austria).

» Product: Oroboros O2k, O2k-Catalogue

O2k-Network Lab: AT Innsbruck Oroboros

Labels: MiParea: Respiration, Instruments;methods 

Organism: Mouse  Tissue;cell: Blood cells  Preparation: Intact cells 

Regulation: Coupling efficiency;uncoupling, Uncoupler  Coupling state: LEAK, ROUTINE, ET  Pathway: ROX  HRR: Oxygraph-2k, TIP2k, O2k-Protocol 

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

Respiration of 32D cells (1·106 ml-1). Traces for the two chambers of the O2k are superimposed. A: Oxygen flow [pmol O2·s-1·106]; B: Oxygen concentration [μM] (graph layout “05b Specific flux overlay”). After inhibition of ATP synthase with oligomycin, FCCP was titrated at 0.5 μM steps with the TIP2k. Each titration step is automatically marked by an event (vertical lines). After the aerobic-anoxic transition, the chambers were opened for re-oxygenation, causing a 10-min disturbance of the traces of respiratory flow. The TIP2k needles were removed before opening, and re-inserted after closing the chamber.

Cell coupling control protocol

DatLab-Analysis templates and DatLab Demo Files

DatLab 7

Calibration-File: MiPNet10.04_CellRespiration_Calib.DLD
Demo-File: MiPNet10.04_CellRespiration.DLD
Chemical Background Excel demo and template: MiPNet10.04_CellRespiration O2k-ChemBackground.xlsx
DatLab-Analysis template: SUIT_MiPNet10.04_CellRespiration.xlsx

Further information

» Gnaiger 2008 POS
» References
» Coupling control protocol
» DatLab