Gnaiger 2019 NatureConference
|Gnaiger E, Passrugger M, Gallee L, Felipe F Garcia-Souza, Zdrazilova L, Doerrier C (2019) High-resolution respirometry and innovations in the Oroboros O2k-technology.|
There is a growing demand for functional analyses of oxidative phosphorylation (OXPHOS analysis) in the areas of mitochondrial physiology and clinical applications, including degenerative diseases and life style-linked preventive medicine. Peripheral blood mononuclear cells (PBMC) and platelets (PLT) are a particularly relevant model for diagnostic OXPHOS analysis.
Over the past 27 years, the Oroboros Oxygraph-2k (O2k) has been established as the unique benchmark instrument for high-resolution respirometry (HRR) of isolated mitochondria, permeabilized and intact cells, tissue preparations and muscle fibres. Accurate measurements are obtained at high sample dilution in a chamber volume of 2 mL. Adherent cells must be detached and suspended for HRR. On the other hand, multi-well systems are designed for high-throughput screening of cellular metabolism in attached cells, which is not the physiological state of blood cells.
Assessment of human mitochondrial function is usually limited by the availability of only small amounts of sample. A blood sample of 9 mL is presently required for two repetitions of an O2k assay with PBMC (4-6 million cells per chamber) and two to three repetitions with platelets (200-250 million cells per chamber). When the volume of the measurement chamber is reduced by a factor of 4, from 2 mL in the O2k to 0.5 mL with the O2k small-volume module (O2k-sV-Module), the required amount of sample per assay is reduced proportionally. The 0.5 mL chamber volume represents a compromise with a focus on monitoring cell respiration in small amounts of sample: (1) Instead of simultaneous measurement of multiple parameters in the O2k-FluoRespirometer, the O2k-sV-Module is designed specifically for respirometry. (2) The O2k is optimized for measurement at deep hypoxia, whereas the O2k-sV-Module is applied in a narrow experimental range of oxygen concentration near air saturation, reducing the technical problems of oxygen diffusion and instrumental O2 background.
Extensive testing of the O2k-sV-Module was completed with intact and permeabilized HEK cells, human platelets and fibroblasts, and with isolated mitochondria, ensuring the quality standards of the Oroboros technology. Substrate-uncoupler-inhibitor-titrations (SUIT) can be applied for OXPHOS phenotyping of mitochondrial respiratory control . The small-volume chamber is suitable for the design of a multi-chamber instrument, for higher sample throughput as required in extensive biomedical studies and large-scale screening of mitochondrial activity.
- Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. - »Bioblast link«
- Erich Gnaiger1,2, Manuela Passrugger1, Leon Gallee1, Luiz Felipe Garcia-Souza1,2, Lucie Zdrazilova1,3, Carolina Doerrier1
- Oroboros Instruments, Schöpfstraße 18, 6020 Innsbruck, Austria
- Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Austria
- First Faculty of Medicine, Charles University, CZ
- This project was supported by K-Regio MitoFit, funded by the Land Tirol within the program K-Regio of Standortagentur Tirol.
- Contribution to COST Action CA15203 MitoEAGLE, supported by COST (European Cooperation in Science and Technology)
- Zdrazilova L was supported by a MitoEAGLE Short-Term Scientific Mission at Oroboros Instruments, AT.
- Garcia-Souza LF was supported by a MitoEAGLE Short-Term Scientific Mission at Lund University, SE.
Labels: MiParea: Respiration
Organism: Human Tissue;cell: Blood cells, HEK, Fibroblast, Platelet Preparation: Intact cells, Permeabilized cells, Isolated mitochondria