O2k Applications

                

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O2k Applications

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

Routine respirometric monitoring with limited activities or amounts of:

• Isolated mitochondria

• Small needle biopsies, permeabilized muscle fibres

• Delicate cell cultures

• Pathological samples with reduced cellular respiration (apoptosis; mitochondrial diseases; ischemia-reperfusion injury, aging, oxidative stress)

• Mutants with diminished respiratory capacity

• Microbial cultures

• Oxygen: at the extremes of intracellular oxygen limitation

• Oxidases and antioxidant systems

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:

• Chamber design: avoiding oxygen-permeable materials such as Perspex or teflon-coated stirrers [7]

• Instrumental concept: including integrated Peltier-temperature control, automatic Titration-Injection microPump TIP2k

• Software features of DatLab

• Methodological standards