Difference between revisions of "Felser 2014 Abstract MiP2014"
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|abstract=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. Ā | |abstract=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. Ā | ||
We analyzed intact cell respiration in Dulbeccoās 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 2.9Ā±0.4 pmol O2.s<sup>-1</sup>.ml<sup>-1</sup> at air saturation. XFe96 background oxygen consumption varied interexperimentally between Ā±3 to Ā±14 pmolāmin<sup>-1</sup>. ROUTINE respiration of two million HSF or HeLa, per O2k chamber, was in the range of 40 pmol O2.s<sup>-1</sup>.ml<sup>-1</sup>. Consistent monolayers of HSF (2.5ā10<sup>4</sup> cells per well) and HeLa (4ā10<sup>4</sup> cells per well) were in the range of 50 and 100 pmolāmin<sup>ā1</sup>, 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). | We analyzed intact cell respiration in Dulbeccoās 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 2.9Ā±0.4 pmol O2.s<sup>-1</sup>.ml<sup>-1</sup> at air saturation. XFe96 background oxygen consumption varied interexperimentally between Ā±3 to Ā±14 pmolāmin<sup>-1</sup>. ROUTINE respiration of two million HSF or HeLa, per O2k chamber, was in the range of 40 pmol O2.s<sup>-1</sup>.ml<sup>-1</sup>. Consistent monolayers of HSF (2.5ā10<sup>4</sup> cells per well) and HeLa (4ā10<sup>4</sup> cells per well) were in the range of 50 and 100 pmolāmin<sup>ā1</sup>, 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). When converted to comparable SI units, ROUTINE oxygen flow was 40 (O2k: HSF and HeLa) versus 33 and 42 (XFe: HSF and HeLa) pmolās<sup>-1</sup>ā10<sup>-6</sup> cells, and ETS was 89 and 67 (O2k: HSF and HeLa) versus 74 and 42 (XFe: HSF and HeLa) pmolās<sup>-1</sup>ā10<sup>-6</sup> cells, respectively. ETS 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. | 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. | ||
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== Conversion to comparable units == | == Conversion to comparable SI units == | ||
O2k (HSF and HeLa): | O2k (HSF and HeLa): 2ā10<sup>6</sup> cells/2 ml yields a cell density of 1ā10<sup>6</sup> cells/ml; volume-specific oxygen flux of 40 pmolās<sup>-1</sup>āml<sup>-1</sup> is then equivalent to ROUTINE oxygen flow per 10<sup>6</sup> cells of 40 pmolās<sup>-1</sup>ā10<sup>-6</sup> cells. At ''R/E''=0.45 and 0.6 in HSF and HeLa, ''E''=89 and 67 pmolās<sup>-1</sup>ā10<sup>-6</sup> cells, respectively. | ||
XFe (HSF and HeLa): 0. | XFe (HSF and HeLa): 0.025ā10<sup>6</sup> (0.04ā10<sup>6</sup>) cells/well and oxygen flow per well of 50 (100) pmolā¢min<sup>-1</sup> or 0.83 (1.7) pmolās<sup>-1</sup> yields ROUTINE oxygen flow per 10<sup>6</sup> cells of 33 (42) pmolās<sup>-1</sup>ā10<sup>-6</sup> cells. At ''R/E''=0.45 and 1.0 in HSF and HeLa, ''E''=74 and 42 pmolās<sup>-1</sup>ā10<sup>-6</sup> cells, respectively. | ||
Ā | |||
Revision as of 10:33, 12 August 2014
Assessing mitochondrial dysfunction in fibroblast cells ā a comparison between O2k and multiwell respirometry. |
Link:
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 Dulbeccoās 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 2.9Ā±0.4 pmol O2.s-1.ml-1 at air saturation. XFe96 background oxygen consumption varied interexperimentally between Ā±3 to Ā±14 pmolāmin-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 (2.5ā104 cells per well) and HeLa (4ā104 cells per well) were in the range of 50 and 100 pmolāminā1, 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). When converted to comparable SI units, ROUTINE oxygen flow was 40 (O2k: HSF and HeLa) versus 33 and 42 (XFe: HSF and HeLa) pmolās-1ā10-6 cells, and ETS was 89 and 67 (O2k: HSF and HeLa) versus 74 and 42 (XFe: HSF and HeLa) pmolās-1ā10-6 cells, respectively. ETS 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.
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 SI units
O2k (HSF and HeLa): 2ā106 cells/2 ml yields a cell density of 1ā106 cells/ml; volume-specific oxygen flux of 40 pmolās-1āml-1 is then equivalent to ROUTINE oxygen flow per 106 cells of 40 pmolās-1ā10-6 cells. At R/E=0.45 and 0.6 in HSF and HeLa, E=89 and 67 pmolās-1ā10-6 cells, respectively.
XFe (HSF and HeLa): 0.025ā106 (0.04ā106) cells/well and oxygen flow per well of 50 (100) pmolā¢min-1 or 0.83 (1.7) pmolās-1 yields ROUTINE oxygen flow per 106 cells of 33 (42) pmolās-1ā10-6 cells. At R/E=0.45 and 1.0 in HSF and HeLa, E=74 and 42 pmolās-1ā10-6 cells, respectively.