Felser 2014 Abstract MiP2014

Assessing mitochondrial dysfunction in fibroblast cells – a comparison between O2k and multiwell respirometry.

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MiP2014, Book of Abstracts Open Access

Felser A, Larsson NG (2014)

Event: MiP2014

Different platforms for the measurement of respiratory fluxes are available. The OROBOROS Oxygraph-2k (O2k) is a two-chamber respirometer based on high-resolution polarographic oxygen sensors. The Seahorse XFe96 extracellular flux analyzer (XFe96) is a fluorescence-based, 96-well sensor cartridge approach. In this study, we aimed to compare both platforms by measuring human skin fibroblasts (HSF) and HeLa cells, which are both characterized by low respiratory fluxes.

We analyzed intact cell respiration in Dulbecco’s Modified Eagle Medium containing pyruvate/ glutamate using a classical phosphorylation control protocol (using oligomycin, CCCP, and rotenone/antimycinA). Instrumental background of the calibrated O2k was 2.9±0.4 pmol O2.s^-1.ml^-1 at air saturation. XFe96 background oxygen consumption varied interexperimentally between ±3 to ±14 pmol∙min^-1. ROUTINE respiration of two million HSF or HeLa, per O2k chamber, was in the range of 40 pmol O2.s^-1.ml^-1. Consistent monolayers of HSF (2.5∙10⁴ cells per well) and HeLa (4∙10⁴ cells per well) were in the range of 50 and 100 pmol∙min⁻¹, respectively. ROUTINE coupling control ratio (ROUTINE flux over electron transfer system capacity) was similar in HSF between both platforms (0.45), whereas the ratio was 1.0 (XFe96) or 0.6 (O2k) in HeLa cells. This difference in HeLa might be influenced by the fact that respiratory flux is assessed in monolayers (XFe96) or suspension (O2k).

In conclusion, O2k and XFe96 are both platforms that are suitable to assess mitochondrial respiration of human fibroblast and HeLa cells. Instrumental background and interexperimental variability is lower in O2k experiments, whereas amount of sample is smaller and throughput of multiple conditions is higher using XFe96 respirometry.

Labels: MiParea: Respiration 

Organism: Human  Tissue;cell: Endothelial; Epithelial; Mesothelial Cell"Endothelial; Epithelial; Mesothelial Cell" is not in the list (Heart, Skeletal muscle, Nervous system, Liver, Kidney, Lung;gill, Islet cell;pancreas;thymus, Endothelial;epithelial;mesothelial cell, Blood cells, Fat, ...) of allowed values for the "Tissue and cell" property.  Preparation: Intact cells 

Regulation: Coupling efficiency;uncoupling  Coupling state: ROUTINE 

HRR: Oxygraph-2k 

MiP2014 

Affiliation

1-Dep Lab Medicine, Karolinska Inst, Stockholm, Sweden; 2-Dep Mitoch Biol, Max Planck Inst Biol Ageing, Cologne, Germany. - andrea.felser@ki.se

Conversion to comparable units

O2k (HSF and HeLa): 2•10⁶ cells/2 ml yields a cell density of 1•10⁶ cells/ml; volume-specific oxygen flux of 40 pmol•s^-1•ml^-1 is then equivalent to ROUTINE oxygen flow per 10⁶ cells of 40 pmol•s^-1•10^-6 cells. At R/E=0.45 and 0.6 in HSF and HeLa, E=89 and 67 pmol•s^-1•10^-6 cells, respectively.

XFe (HSF and HeLa): 0.025•10⁶ (0.04•10⁶) cells/well and oxygen flow per well of 50 (100) pmol•min^-1 or 0.83 (1.7) pmol•s^-1 yields ROUTINE oxygen flow per 10⁶ cells of 33 (42) pmol•s^-1•10^-6 cells. At R/E=0.45 and 1.0 in HSF and HeLa, E=74 and 42 pmol•s^-1•10^-6 cells, respectively.

ETS capacity in HSF and HeLa was approximately 80% and 60% in the XFe compared to the O2k.