Membrane Potential Changes Visualized in Complete Growth Media through Confocal Laser Scanning Microscopy of bis-Oxonol-Loaded Cells

Confocal laser scanning microscopy (CLSM) was employed to visualize and measure membrane potential changes in several types of cultured adherent cells, such as human fibroblasts, mouse mammary tumor C127 cells, and human saphenous vein endothelial cells, preloaded with the anionic dye bis-1,3,-diethylthiobarbituratetrimethineoxonol (bis-oxonol). The fluorescence of cell-associated bis-oxonol was detected in a single confocal plane. An original flow-chamber apparatus was employed to replace the extracellular medium, avoiding alterations of the plane selected for observation. In all the cell types and the experimental situations tested the intracellular distribution of the dye was typical; perinuclear zones accumulated the dye which, conversely, was excluded by the nucleus. Fluorescence was calibrated versus the membrane potential by varying the extracellular concentration of sodium in the presence of gramicidin. With this approach membrane potential was measured (i) in cultured human fibroblasts incubated under anisotonic conditions, (ii) in heterogeneous cell populations which respond unevenly to potential perturbing conditions, and (iii) in human macrovascular endothelial cells maintained in high-serum, complete growth medium. The results obtained indicate that CLSM can be successfully employed to measure changes of membrane potential in single, bis-oxonol-loaded adherent cells under experimental conditions which severely hinder conventional spectrofluorimetric approaches.