MiPNet12.11 MitoRespiration

Hand SC, Lemieux H, Gnaiger E (2007) Flux control ratios in isolatd mitochondria. OXPHOS capacity and respiratory control in isolated mitochondria. Mitochondr Physiol Network 12.11.

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OROBOROS (2007) Mitochondr Physiol Network

Abstract: [[has abstract::O2k-Protocols for Basic Protocol with Isolated Mitochondria: LEAK, OXPHOS, ETS, ROX.

Methodological and conceptual features of high-resolution respirometry are illustrated in an experiment with isolated mitochondria in the OROBOROS Oxygraph-2k (O2k). The experiment demonstrates manual titrations applied to study respiratory control ratios and respiratory capacity in isolated mitochondria. Application of the DatLab 4.2 software is shown for on-line data analysis. The following guideline describes the experimental protocol and includes a short discussion of results. The experiments were carried out by participants of an O2k-Course on HRR in December 2006 (IOC36; Schröcken, Austria).

Figure 1. Experiment 2006-12-14 CD-03.DLD

The protocol (Fig. 1) was developed for respiratory studies of isolated mitochondria, and has general implications on critically assessing experiments with isolated mitochondria or permeabilized cells and tissues. As a substrate, succinate+rotenone or succinate without rotenone was chosen in the present example (MiPNet11.09). The experiment was aimed at assessing (1) respiratory OXPHOS and LEAK capacity, in the coupled ADP activated state and LEAK state after phosphorylation of ADP to ATP (States P and L; check for possible limitations by non-saturating ADP-concentrations or loss of cytochrome c), (2) capacity of the electron transport system (ETS) in the uncoupled state (State E; FCCP titration, avoiding inhibition by high FCCP concentrations), and (3) coupling control of OXPHOS (underestimated by the conventional RCR if the phosphorylation system is limiting).

>> O2k-Protocols:Overall contents

>> Product: OROBOROS Oxygraph-2k, O2k-Catalogue]]

• O2k-Network Lab: AT_Innsbruck_OROBOROS

Labels: MiParea: Respiration, Instruments;methods 

Organism: Artemia 

Preparation: Isolated Mitochondria 

Regulation: Coupling efficiency;uncoupling  Coupling state: LEAK, OXPHOS, ETS 

HRR: Oxygraph-2k, Protocol 

O2k-Demo 

Further information

>>Excel Templates, DatLab Templates and DatLab-Demo Files

>> MiPNet12.11

• Version 5: 2011-12-11
• Version 1: 2007

• References