Gnaiger 2019 NatureConference

Gnaiger E, Passrugger M, Gallee L, Felipe Garcia-Souza L, Zdrazilova1 L, Doerrier C (2019) High-resolution respirometry and innovations in the Oroboros O2k technology.

Link: Nature Conference 2019 Xiamen CN

Gnaiger E, Passrugger M, Gallee L, Felipe Garcia-Souza L, Zdrazilova1 L, Doerrier C (2019)

Event: Nature Conference 2019 Xiamen CN

There is a growing demand for functional analyses of oxidative phosphorylation (OXPHOS analysis) in the areas of mitochondrial biology and clinical applications, including degenerative diseases and life style-linked preventive medicine. Peripheral blood mononuclear cells (PBMCs) and platelets are a particularly relevant model for such diagnostic studies.

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 O2k chambers with PBMCs (4-6 million cells per chamber) and two to three O2k chambers 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 in the small chamber respirometer (SCR), the required amount of sample per assay is reduced proportionally. The small chamber volume represents a compromise with a focus on the monitoring of cell respiration in small amounts of sample: (1) Instead of simultaneous monitoring of multiple parameters in the O2k-FluoRespirometer, the SCR is designed for respirometry only. (2) The O2k is optimized for measurement at deep hypoxia, whereas the SCR 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 SCR 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 [1]. The small chamber is suitable for the design of a multi-chamber instrument, for high sample throughput as required in extensive biomedical studies and large-scale screening of mitochondrial activity.

1. 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«

• Bioblast editor: Gnaiger E • O2k-Network Lab: AT Innsbruck Oroboros

Labels: MiParea: Respiration 

Organism: Human  Tissue;cell: Blood cells, HEK, Fibroblast, Platelet  Preparation: Intact cells, Permeabilized cells, Isolated mitochondria 

HRR: Oxygraph-2k