Comparison of the effect of mitochondrial inhibitors on mitochondrial membrane potential in two different cell lines using flow cytometry and spectrofluorometry

Abstract

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.

Conclusion: 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.