High-Resolution Respirometry (HRR): O2k-Applications

  • Diagnosis of acquired and genetic mitochondrial diseases
  • Diagnosis of myopathies and neuromuscular pathologies
  • Studies on cell function and cell death - apoptosis and necrosis 
  • Oxidative stress 
  • Ischemia-reperfusion injury
  • Aging and senescence 
  • Pharmacological tests

 For more applications, browse through the O2k-Publications



High-Resolution: high accuracy, minimum amount of sample
 
 
 
 
 
Sample 
Amount 
O2k-Chamber 
Temp. 
 
 
 
 
 
 
Heart mitochondria 
0.01 mg protein 
2.0 ml 
37 C 
 
Permeabilized muscle fibers 
1 mg wet weight 
2.0 ml 
37 C 
 
Endothelial cells 
0.2 10^6 cells 
2.0 ml 
37 C 
 
T-lymphocytes 
1 10^6 cells  
2.0 ml 
37 C 
 


Reference: Pesta 2012 Methods Mol Biol



High-Resolution Respirometry


Figure modified after: Gnaiger E., Lassnig B., Kuznetsov A.V., Rieger G., Margreiter R. (1998) Mitochondrial oxygen affinity, respiratory flux control and excess capacity of cytochrome c oxidase. J. exp. Biol. 201: 1129-1139.

Routine respirometric monitoring with limited activities or amounts of:



Rapidly Increasing Demands

 

Approaches to mitochondrial and cellular respirometry have been transformed and extended in mitochondrial physiology, from specialized bioenergetics to integrative and clinical applications. Meeting rapidly increasing demands, the new OROBOROS Oxygraph-2k (O2k) covers major areas of interest:

  1. Routine analysis of oxidative phosphorylation (OXPHOS) in diverse fields of research such as redox signaling, oxidative stress [1], ischemia-reperfusion [2], aging [3], degenerative and inherited diseases [4]; apoptosis, cancer research and drug testing [5], cell signaling [6], and environmental stress. For the non-specialist, the O2k provides robustness and reliability of instrumental performance. Full software support by DatLab includes automatic oxygen calibration, continuous display of oxygen concentration and respiratory flux, and on-line tabulation of final results on OXPHOS activity during selected experimental sections. Standardized O2k quality controls eliminate interferences by instrumental artifacts. Long-term stability of the polarographic oxygen sensors ensures the O2k to be ready to use (no exchange of membranes and electrolyte for months).
  2. High throughput is required in research with cell cultures and pharmacological tests. The O2k has two independent chambers for parallel experimental runs. User-friendly features make it possible to apply several O2k simultaneously.
  3. Diagnosis of OXPHOS in living and permeabilized cells, small amounts of tissue from transgenic animal models, and human needle biopsies requires high-resolution respirometry to obtain accurate results even at high sample dilution. <1 mg fresh muscle tissue or <500.000 cells are required per chamber (10-fold less compared to conventional instruments). Multiple substrate/inhibitor titration protocols extend the information gained on various OXPHOS complexes in a single incubation.
  4. Prevalence of oxidative stress at air-level oxygen tensions demands a shift to physiological low-oxygen ranges for studying OXPHOS [7, 8]. 
  5. O2k-MultiSensor applications (pH, NO, H2O2, ion sensitive electrodes), and sampling from the chamber (CS enzyme assays; EPR spin probes for ROS) extend the diagnostic power of high-resolution respirometry.

This makes the OROBOROS Oxygraph-2k the world-wide unique instrument for high-resolution respirometry:



Key references introducing HRR

 

  • Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58. >>Abstract
  • Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley: 327-352. >>Abstract
  • Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128: 277-297. >>Abstract


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OROBOROS High-Resolution Respirometry