EXPERIMENTAL CELL RESEARCH 231, 260 –268 (1997) ARTICLE NO. EX963469 Membrane Potential Changes Visualized in Complete Growth Media through Confocal Laser Scanning Microscopy of bis-Oxonol-Loaded Cells VALERIA DALL’ASTA,RITA GATTI,* GUIDO ORLANDINI,† PATRIZIA A. ROSSI,BIANCA M. ROTOLI, ROBERTO SALA,OVIDIO BUSSOLATI, AND GIAN C. GAZZOLA 1 Institutes of General Pathology, *Anatomy, and †Clinical Medicine — Nephrology, Universita ` degli Studi di Parma, I-43100 Parma, Italy [1, 2]. Confocal laser scanning microscopy (CLSM) has Confocal laser scanning microscopy (CLSM) was em- made possible a direct, dynamic imaging of these pa- ployed to visualize and measure membrane potential rameters in living adherent cells. Among these param- changes in several types of cultured adherent cells, eters, membrane potential has also been evaluated such as human fibroblasts, mouse mammary tumor with CLSM in cells and tissues [3 –7]. Most of those C127 cells, and human saphenous vein endothelial studies employ positively charged dyes [3 – 6] which are cells, preloaded with the anionic dye bis-1,3,-dieth- accumulated in the mitochondria. Thus, while these ylthiobarbituratetrimethineoxonol (bis-oxonol). The probes allow a simultaneous analysis of membrane po- fluorescence of cell-associated bis-oxonol was detected tential across both plasma membrane and mitochon- in a single confocal plane. An original flow-chamber drial membrane, the measure of plasma membrane po- apparatus was employed to replace the extracellular tential can be complicated by signals of mitochondrial medium, avoiding alterations of the plane selected for origin which often overwhelm those derived from cyto- observation. In all the cell types and the experimental plasm [6]. Moreover, a careful evaluation of metabolism situations tested the intracellular distribution of the and morphology of dye-loaded cells is required to ex- dye was typical; perinuclear zones accumulated the clude possible toxic effects due to the high intramito- dye which, conversely, was excluded by the nucleus. chondrial accumulation of positively charged dyes [8]. Fluorescence was calibrated versus the membrane po- On the contrary, negative-charged dyes, such as bis- tential by varying the extracellular concentration of oxonol, are excluded by mitochondria and avoid such sodium in the presence of gramicidin. With this ap- complications [9]. Nevertheless, a recent attempt to proach membrane potential was measured (i) in cul- employ this dye in CLSM [7] has only yielded qualita- tured human fibroblasts incubated under anisotonic tive data and failed to reach a quantitative assessment conditions, (ii) in heterogeneous cell populations of the membrane potential. which respond unevenly to potential perturbing con- Bis-oxonol slowly distributes across biological mem- ditions, and (iii) in human macrovascular endothelial cells maintained in high-serum, complete growth me- branes according to the membrane potential and binds dium. The results obtained indicate that CLSM can be to hydrophobic cell components; since the quantum successfully employed to measure changes of mem- yield of the dye increases impressively upon the bind- brane potential in single, bis-oxonol-loaded adherent ing, the fluorescence of cells incubated in a medium cells under experimental conditions which severely containing bis-oxonol increases upon depolarization hinder conventional spectrofluorimetric approaches. and, conversely, decreases with hyperpolarization [9].  1997 Academic Press The traditional technique employed for bis-oxonol mea- surements has been fluorimetry with both suspended and, more recently, adherent cells [10 – 12]. However, INTRODUCTION bis-oxonol spectrofluorimetry is affected by several shortcomings: (i) no inspection of the intracellular dye An increasing number of fluorescent indicators are distribution, which could be perturbed by the experi- available for a variety of cell functional parameters mental manipulation, is possible, (ii) the overall signal derives also from the probe present in the extracellular 1 To whom correspondence and reprint requests should be ad- compartment, which is often 2–3 orders of magnitude dressed at the Istituto di Patologia Generale, Universita ` di Parma, larger than the intracellular space, (iii) serum must be Via Gramsci, 14, I-43100 Parma, Italy. Fax: /39 521 980388; E-mail: gazzola@ipruniv.cce.unipr.it. absolutely omitted from extracellular medium to avoid 260 0014-4827/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.