Krumschnabel 2016 Abstract Mito Xmas Meeting Innsbruck
|Establishment of a reference sample for high-resolution respirometry.|
Mitochondrial respiratory function may be assessed and analyzed in great detail applying high-resolution respirometry (HRR), which is thus a valuable tool for the diagnostic assessment of function and dysfunction of oxidative phosphorylation related to a wide range of metabolic and other diseases. In order to allow for a valid comparison of data among research groups it appears necessary to agree on certain standards relating to sample treatment and measurements protocols, but an ultimate tool for data validation and comparison would be obtained with an available common references sample. Here, we report on efforts on establishing such a reference sample, which is functionally stable over time and can be distributed across the globe to other labs and could provide the basis of standard proficiency tests within and between reference laboratories. We chose a widely applied human cell line, HEK293T cells, which can be examined using reference substrate-uncoupler-inhibitor titration (SUIT) protocols, and evaluated conditions for cell storage for later and reproducible respirometric assessment. We found that after storage of these cells in standard freeze medium at -80°C cell viability remained largely preserved and that respirometric parameters showed an initial decrease in the first two weeks of storage but remained fairly constant thereafter for 3, but not for 6 month. Storing already permeabilized cells led to even better preservation of respiratory function for at least 3 month. First experiments after storage of cells in dry ice for 48h indicated similarly preserved mitochondrial function and altogether these results suggest that HEK293T cells may be regarded promising candidates for a respirometric reference sample.
Labels: MiParea: Respiration, Instruments;methods
Stress:Cryopreservation Organism: Human Tissue;cell: HEK Preparation: Intact cells, Permeabilized cells
HRR: Oxygraph-2k Event: Poster
- Oroboros Instruments, Innsbruck, Austria