Fluorescent probing of membrane potential in walled cells: diS‐C 3 (3) assay in Saccharomyces cerevisiae

Membrane‐potential‐dependent accumulation of diS‐C 3 (3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, λ max , caused by a readily reversible probe binding to cell constituents. Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift. As the potential‐reporting parameter, the λ max shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity. The magnitude of the potential‐dependent red λ max shift depends on relative cell‐to‐probe concentration ratio, a maximum shift (572→582 nm) being found in very thick suspensions and in cell lysates. The potential therefore has to be assessed at reasonably low cell (≤5×10 6 cells/ml) and probe (10 −7 M) concentrations at which a clearly defined relationship exists between the λ max shift and the potential‐dependent accumulation of the dye in the cells. The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10–30 min because the cell wall acts as a barrier, hampering probe penetration into the cells. The barrier properties of the cell wall correlate with its thickness: cells grown in 0·2% glucose (cell wall thickness 0·175±0·015 μm, n =30) are stained much faster and the λ max is more red‐shifted than in cells grown in 2% glucose (cell wall thickness 0·260±0·043 μm, n =44). At a suitable cell and probe concentration and under standard conditions, the λ max shift of diS‐C 3 (3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae . © 1998 John Wiley & Sons, Ltd.