Felser 2014 Abstract MiP2014
|Assessing mitochondrial dysfunction in fibroblast cells – a comparison between O2k and multiwell respirometry.|
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-pathway capacity, ET-pathway) 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). When converted to comparable SI units, ROUTINE oxygen flow was 40 (O2k: HSF and HeLa) versus 33 and 42 (XFe: HSF and HeLa) pmol∙s-1∙10-6 cells, and ET-pathway was 89 and 67 (O2k: HSF and HeLa) versus 74 and 42 (XFe: HSF and HeLa) pmol∙s-1∙10-6 cells, respectively. ET capacity in HSF and HeLa was approximately 80% and 60% in the XFe compared to the 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.
• O2k-Network Lab: DE Cologne Larsson NG
Labels: MiParea: Respiration
Organism: Human Tissue;cell: Endothelial;epithelial;mesothelial cell, HeLa, Fibroblast Preparation: Intact cells
Regulation: Coupling efficiency;uncoupling Coupling state: ROUTINE, ET Pathway: ROX HRR: Oxygraph-2k Event: B4, Oral MiP2014
1-Dep Lab Medicine, Karolinska Inst, Stockholm, Sweden; 2-Dep Mitoch Biol, Max Planck Inst Biol Ageing, Cologne, Germany. - [email protected]
Conversion to comparable SI 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.