MEMBRANE-POTENTIAL CHANGES VISUALIZED IN COMPLETE GROWTH MEDIA THROUGH CONFOCAL LASER-SCANNING MICROSCOPY BIS-OXONOL-LOADED CELLS

Confocal laser scanning microscopy (CLSM) was employed to visualize an d measure membrane potential changes in several types of cultured adhe rent, cells, such as human fibroblasts, mouse mammary tumor C127 cells , and human saphenous vein endothelial cells, preloaded with the anion ic dye bis-1,3,-diethylthiobarbituratetrimethineoxonol (bis-oxonol). T he fluorescence of cell-associated bis-oxonol was detected in a single confocal plane, An original flow-chamber apparatus was employed to re place the extracellular medium, avoiding alterations of the plane sele cted for observation. in all the cell types and the experimental situa tions tested the intracellular distribution of the dye was typical; pe rinuclear zones accumulated the dye which, conversely, was excluded by the nucleus. Fluorescence was calibrated versus the membrane potentia l 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 poten tial perturbing conditions, and (iii) in human macrovascular endotheli al cells maintained in high-serum, complete growth medium. The results obtained indicate that CLSM can be successfully employed to measure c hanges of membrane potential in single, bis-oxonol-loaded adherent cel ls under experimental conditions which severely hinder conventional sp ectrofluorimetric approaches. (C) 1997 Academic Press.