Kalbacova 2003 Cytometry: Difference between revisions
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|abstract=Background: Determination of mitochondrial membrane | |abstract=Background: Determination of mitochondrial membrane | ||
potential ((m) is widely used to characterize cellular | potential ((m) is widely used to characterize cellular | ||
metabolism, viability, and apoptosis. Changes of | metabolism, viability, and apoptosis. Changes of ฮฮจm | ||
induced by inhibitors of oxidative phosphorylation characterize | induced by inhibitors of oxidative phosphorylation characterize | ||
respective contributions of mitochondria and glycolysis | respective contributions of mitochondria and glycolysis | ||
to adenosine triphosphate (ATP) synthesis. | to adenosine triphosphate (ATP) synthesis. | ||
Methods: | Methods: ฮฮจm in BSC-40 and HeLa G cell lines was | ||
determined by flow cytometry and spectrofluorometry. Its | determined by flow cytometry and spectrofluorometry. Its | ||
changes induced by specific mitochondrial inhibitors | changes induced by specific mitochondrial inhibitors | ||
were evaluated using 3, | were evaluated using 3,3ฮฮจ-dihexyloxacarbocyanine iodide | ||
(DiOC6(3)), tetramethylrhodamine ethyl ester, and Mito- | (DiOC6(3)), tetramethylrhodamine ethyl ester, and Mito- | ||
Tracker Red. Mitochondrial function was further characterized | Tracker Red. Mitochondrial function was further characterized | ||
by oxygen consumption. | by oxygen consumption. | ||
Results: Inhibition of respiration by antimycin A or uncoupling | Results: Inhibition of respiration by antimycin A or uncoupling | ||
of mitochondria by FCCP decreased | of mitochondria by FCCP decreased ฮฮจm in | ||
both cell lines. Inhibition of ATP production by oligomycin | both cell lines. Inhibition of ATP production by oligomycin | ||
or atractyloside induced a moderate decrease of | or atractyloside induced a moderate decrease of ฮฮจm | ||
in HeLa G cells and an increase of | in HeLa G cells and an increase of ฮฮจm in BSC-40 cells. | ||
Statistically significant differences in | Statistically significant differences in ฮฮจm between the | ||
two cell lines were found with both flow cytometry and | two cell lines were found with both flow cytometry and | ||
spectrofluorometry. Respirometry showed higher basal | spectrofluorometry. Respirometry showed higher basal | ||
and FCCP-stimulated respiration in BSC-40 cells. | and FCCP-stimulated respiration in BSC-40 cells. | ||
Conclusion: Changes of | Conclusion: Changes of ฮฮจm and oxygen consumption | ||
showed that BSC-40 cells are more sensitive than HeLa G | showed that BSC-40 cells are more sensitive than HeLa G | ||
cells to inhibitors of mitochondrial function, suggesting | cells to inhibitors of mitochondrial function, suggesting | ||
that BSC-40 cells are more dependent than HeLa G cells on | that BSC-40 cells are more dependent than HeLa G cells on | ||
aerobic ATP production. Determination of | aerobic ATP production. Determination of ฮฮจm changes | ||
by flow cytometry exhibited greater sensitivity than the | by flow cytometry exhibited greater sensitivity than the | ||
ones by spectrofluorometry. Cytometry Part A 52A: | ones by spectrofluorometry. Cytometry Part A 52A: | ||
Revision as of 16:23, 4 October 2010
| Kalbacova M, Vrbacky M, Drahota Z, Melkova Z (2003) Comparison of the effect of mitochondrial inhibitors on mitochondrial membrane potential in two different cell lines using flow cytometry and spectrofluorometry. Cytometry 52A: 110-116. |
Kalbacova M, Vrbacky M, Drahota Z, Melkova Z (2003)
Abstract: Background: Determination of mitochondrial membrane potential ((m) is widely used to characterize cellular metabolism, viability, and apoptosis. Changes of ฮฮจm induced by inhibitors of oxidative phosphorylation characterize respective contributions of mitochondria and glycolysis to adenosine triphosphate (ATP) synthesis. Methods: ฮฮจm in BSC-40 and HeLa G cell lines was determined by flow cytometry and spectrofluorometry. Its changes induced by specific mitochondrial inhibitors were evaluated using 3,3ฮฮจ-dihexyloxacarbocyanine iodide (DiOC6(3)), tetramethylrhodamine ethyl ester, and Mito- Tracker Red. Mitochondrial function was further characterized by oxygen consumption. Results: Inhibition of respiration by antimycin A or uncoupling of mitochondria by FCCP decreased ฮฮจm in both cell lines. Inhibition of ATP production by oligomycin or atractyloside induced a moderate decrease of ฮฮจm in HeLa G cells and an increase of ฮฮจm in BSC-40 cells. Statistically significant differences in ฮฮจm between the two cell lines were found with both flow cytometry and spectrofluorometry. Respirometry showed higher basal and FCCP-stimulated respiration in BSC-40 cells. Conclusion: Changes of ฮฮจm and oxygen consumption showed that BSC-40 cells are more sensitive than HeLa G cells to inhibitors of mitochondrial function, suggesting that BSC-40 cells are more dependent than HeLa G cells on aerobic ATP production. Determination of ฮฮจm changes by flow cytometry exhibited greater sensitivity than the ones by spectrofluorometry. Cytometry Part A 52A: 110โ116, 2003.
Labels:
Stress:Cancer; Apoptosis; Cytochrome c"Cancer; Apoptosis; Cytochrome c" is not in the list (Cell death, Cryopreservation, Ischemia-reperfusion, Permeability transition, Oxidative stress;RONS, Temperature, Hypoxia, Mitochondrial disease) of allowed values for the "Stress" property.
Tissue;cell: Blood Cell; Suspension Culture"Blood Cell; Suspension Culture" 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.
Regulation: Respiration; OXPHOS; ETS Capacity"Respiration; OXPHOS; ETS Capacity" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Coupling; Membrane Potential"Coupling; Membrane Potential" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Ion Homeostasis"Ion Homeostasis" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Substrate; Glucose; TCA Cycle"Substrate; Glucose; TCA Cycle" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property.
HRR: Oxygraph-2k, Spectrophotometry; Spectrofluorimetry"Spectrophotometry; Spectrofluorimetry" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.
