Pranger 2018 Emerging Concepts in Mitochondrial Biology

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High-resolution respirometry with a reduced chamber volume – which are the compromises in OXPHOS analysis?

Link: [1]

Pranger F, Gnaiger E (2018)

Event: Emerging Concepts in Mitochondrial Biology

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

Over the past 25 years, the Oroboros 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 need to be trypsinized and suspended for HRR. 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 sample availability. A blood sample of 9 mL is 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 chamber volume is reduced by a factor of 4, to 0.5 mL in the small-chamber respirometer (O2k-SCR), the required amount of sample per assay can be reduced proportionally. The 0.5 mL volume represents a compromise, focusing on cell respiration with small amounts of sample: (1) Instead of simultaneous monitoring of multiple parameters in the O2k-FluoRespirometer, the O2k-SCR is designed for respirometry only. (2) The O2k-FluoRespirometer is optimized for measurements from deep hypoxia to hyperoxia, whereas the O2k-SCR is applied near air saturation, reducing technical problems of oxygen diffusion and instrumental O2-background.

Extensive testing of the O2k-SCR is in progress to ensure the high quality standards of the Oroboros technology. The reduced volume facilitates the design of a multi-chamber instrument for high sample throughput as required in extensive biomedical studies and large-scale screening of mitochondrial activity.


Bioblast editor: Kandolf G, Pranger F O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E


Labels: MiParea: Respiration, Instruments;methods 


Tissue;cell: Blood cells, Platelet 



HRR: Oxygraph-2k 

PBMCs 

Affiliations

Oroboros Instruments, Schöpfstraße 18, 6020 Innsbruck, Austria. - florian.pranger@oroboros.at