|Gnaiger E, Gradl P (2007) Oxygraph-2k: Paradigm shift and features of high-resolution respirometry. Mitochondr Physiol Network 09.01.|
Abstract: Modern trends in mitochondrial physiology and mitochondrial respiratory pathology set advanced standards, and present new requirements with respect to high-resolution respirometry of isolated mitochondria, cultured cells, tissue preparations and human biopsies. For more than ten years, the OROBOROS O2k is being referred to as the unique instrumental system for high-resolution respirometry. The new OROBOROS O2k continues this well appreciated tradition, and extends the experimental options on the basis of chamber design, electronics and the task-specific DatLab software. The OROBOROS O2k combines the power of a user-friendly scientific strategy with the skill of professional hardware and software development. The O2k reinforces the scientific strength of an international network dedicated to apply this unique instrument at its best in fundamental science and biomedical research. »Versions
- Version 1: 2007
- Version 5: 2011-12-03
• O2k-Network Lab: AT_Innsbruck_OROBOROS
HRR: Oxygraph-2k, TIP2k
- O2k-Chamber: Paradigm shift in HRR
- The glass chamber and PVDF stirrer of the Oxygraph-2k.
- Oxygraph-2k: Paradigm shift and features of high-resolution respirometry. MiPNet09.01. Including sole source information. Bioblast Link
- Small chamber volumes of 20-50 µl generate problems rather than providing resolution with small amounts of tissue samples, low numbers of cultured cells or few isolated mitochondria. The surface to volume ratio increases with decreasing chamber volume, thus various boundary effects entail larger errors at smaller volume, in particular oxygen diffusion. While the rate of oxygen depletion per unit amount of sample increases linearly with decreasing chamber volume, side effects may increase to a larger degree. Then, sensitivity is lost with decreasing chamber size. We recommend a minimum chamber volume of 1.5-2.0 cm3.
- Paradigm shift from mimimum to optimum chamber volume
- 1. Minimization of chamber volume represents a past paradigm, aming at high rates of oxygen consumption per volume. The advantage appears to be obvious, whereas the drawbacks are frequently overlooked (see below).
- 2. Advancements of electronics, data acquisition and analysis, polarographic oxygen sensor specifications and chamber design made possible a superior approach, allowing for respirometric measurements at high dilution, as reviewed by Gnaiger E (2001). In specifically designed mitochondrial respiration media, respiration is stable at high dilution, multiple substrate/inhibitor titrations are possible without oxygen depletion, and a low-oxygen regime may be chosen to prevent elevation of oxidative stress at air-level oxygen saturation. In contrast, micro-chambers are characterized by a high surface-to-volume ratio which hinders optimum stirring, increases unfavourable surface effects and oxygen-backdiffusion, and poses problems with accurate titrations and dilution effects of the sample. These potential - and mostly hidden - artefacts are avoided in high-resolution respirometry, using glass chambers, titanium stoppers, and avoiding teflon-coated stirrers or perspex (yielding high back-diffusion of oxygen).
- Assume you have 0.1 mg mitochondrial protein for a respirometric assay. Approach (1) would lead you to search for a 100 µl volume respirometer, to maintain a classical 1 mg/ml protein concentration. In contrast, high-resolution respirometry allows for dilution of mitochondria to 0.02 mg/ml protein. Dilution of 0.1 mg mitochondrial protein in a 2 ml chamber yields an optimum concentration for multiple substrate/inhibitor titrations and kinetic measurements. The high-resolution approach of the OROBOROS Oxygraph-2k offers the unique advantages of a versatile and ready-to-use system for studies in mitochondrial physiology and pathology.