Membrane Potential Estimation by Flow Cytometry

Abstract

Membrane potential (ΔΨ) is generated and maintained by concentration gradients of ions such as sodium, potassium, chloride, and hydrogen. Changes in cytoplasmic ΔΨ in the course of surface-receptor-mediated processes related to the development, function, and pathology of many cell types often play a role in transmembrane signaling. Cytoplasmic ΔΨ is also reduced to zero when the membrane is ruptured by chemical or physical agents. Mitochondrial ΔΨ is reduced when energy metabolism is disrupted, notably in apoptosis. In bacteria, which lack mitochondria, ΔΨ reflects both the state of energy metabolism and the physical integrity of the cytoplasmic membrane. Flow cytometry can be used to estimate membrane potential in eukaryotic cells, mitochondria in situ, isolated mitochondria, and bacteria. Older methods, using lipophilic cationic dyes such as the cyanines and rhodamine 123 or lipophilic anionic dyes such as the oxonols can detect relatively large changes in ΔΨ and identify heterogeneity of response in subpopulations comprising substantial fractions of a cell population. Newer ratiometric techniques allow precise measurement of ΔΨ to within 10 mV or less. Among other factors, action of efflux pumps, changes in membrane structure, and changes in protein or lipid concentration in the medium in which cells are suspended can produce changes in cellular fluorescence which may be misinterpreted as changes in ΔΨ. Techniques for estimation and measurement of ΔΨ therefore typically require careful control of cell and reagent concentrations and incubation times and selection of appropriate controls if they are to provide accurate information.