Unbiased Millivolts Assay of Mitochondrial Membrane Potential in Intact Cells

The Reduction in the Mitochondrial Membrane Potential in Aging: The Role of the Mitochondrial Permeability Transition Pore

“…The dye concentration used in these studies was very high (26 µM), orders of magnitude higher than the recommended dye concentrations needed to avoid aggregation and quenching. It was previously demonstrated that only when the external concentration of the dye is in the low nM range is the fluorescence of the dye in the mitochondrial matrix not quenched, and the magnitude of ∆Ψm is proportional to the fluorescence intensity [39][40][41]. Therefore, it is quite clear that, in the experiments of Haggen et al [48][49][50], the fluorescence intensity is saturated and, therefore, insensitive to the magnitude of ∆Ψm.…”

“…In this method, it is necessary to use very low dye concentrations to prevent aggregation and quenching in the mitochondrial matrix since this would saturate the fluorescence and the cell fluorescence would no longer be proportional to ∆Ψm. Other factors also need to be considered, e.g., the mitochondrial content of the cells (which may be reduced in aging), the mitochondrial matrix volume, and the plasma membrane potential that drives the accumulation of the dye into the cell [39][40][41]. The fluorescence maximum of the lipophilic cation JC-1 shifts from green fluorescence of the monomer to red fluorescence of the aggregated dye, and, therefore, ∆Ψm is estimated from the ratio of red/green fluorescence [42].…”

The Reduction of Mitochondrial Membrane Potential in Aging: The Role of the Mitochondria Permeability Transition Pore

“…The dye concentration used in these studies was very high (26 uM), orders of magnitude higher than the recommended dye concentrations needed to avoid aggregation and quenching. It was previously demonstrated that only when the external concentration of the dye is in the low nM range the fluorescence of the dye in the mitochondrial matrix is not quenched and the magnitude of ∆Ψm is proportional to the fluorescence intensity [39][40][41]. Therefore, it is quite clear that in the experiments of Haggen et al [48][49][50] the fluorescence intensity is saturated and therefore insensitive to the magnitude of ∆Ψm .…”

“…In this method it is necessary to use very low dye concentrations to prevent aggregation and quenching in the mitochondrial matrix since that would saturate the fluorescence and the cell fluorescence would no longer be proportional to ∆Ψm. Other factors also needed to be considered, e.g., the mitochondrial content of the cells (which may be reduced in aging), the mitochondrial matrix volume, and the plasma membrane potential that drive the accumulation of the dye into the cell [39][40][41]. The fluorescence maximum of the lipophilic cation JC-1 shifts from green fluorescence of the monomer to red fluorescence of the aggregated dye, and therefore ∆Ψm can be estimated from the ratio red/green fluorescence [42].…”