Comparison of the effect of mitochondrial inhibitors on mitochondrial membrane potential in two different cell lines using flow cytometry and spectrofluorometry.
BACKGROUND
Determination of mitochondrial membrane potential (DeltaPsim) is widely used to characterize cellular metabolism, viability, and apoptosis. Changes of DeltaPsim induced by inhibitors of oxidative phosphorylation characterize respective contributions of mitochondria and glycolysis to adenosine triphosphate (ATP) synthesis.


METHODS
DeltaPsim 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 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)), tetramethylrhodamine ethyl ester, and MitoTracker Red. Mitochondrial function was further characterized by oxygen consumption.


RESULTS
Inhibition of respiration by antimycin A or uncoupling of mitochondria by FCCP decreased DeltaPsim in both cell lines. Inhibition of ATP production by oligomycin or atractyloside induced a moderate decrease of DeltaPsim in HeLa G cells and an increase of DeltaPsim in BSC-40 cells. Statistically significant differences in DeltaPsim 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.


CONCLUSIONS
Changes of DeltaPsim 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 DeltaPsim changes by flow cytometry exhibited greater sensitivity than the ones by spectrofluorometry.
Kalbácová
Marie
M
Department of Pathological Physiology, 1st Medical Faculty, Charles University, Prague, Czech Republic.
Vrbacký
Marek
M
Drahota
Zdenek
Z
Melková
Zora
Z
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D001278:Atractyloside / Q000494:pharmacology; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002522:Chlorocebus aethiops; D005434:Flow Cytometry / Q000379:methods*; D006367:HeLa Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D009840:Oligomycins / Q000494:pharmacology; D010101:Oxygen Consumption / Q000187:drug effects; D013053:Spectrophotometry / Q000379:methods*; D014475:Uncoupling Agents / Q000494:pharmacology
D009840:Oligomycins; D014475:Uncoupling Agents; D001278:Atractyloside; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000968:Antimycin A