FLOW CYTOMETRIC DETERMINATION OF ABSOLUTE MEMBRANE-POTENTIAL OF CELLS

Membrane potential measurements using fluorescent membrane potential i ndicator dyes report on relative changes but usually do not result in an absolute value of the measured parameter. The method developed in t his paper is based on the assumption that the negatively charged bis-o xonol distributes across the cytoplasmic membrane according to the Ner nst equation. It is further supposed that the fluorescence intensity m easured from a given stained cell is a single-value function of the in tracellular dye concentration. The protocol suggested incorporates the construction of a calibration curve (fluorescence intensity measured from stained cells vs. extracellular dye concentration). This allows t he evaluation of the membrane potential in millivolts using fluorescen ce readings of the cells both in the depolarized state and in the stat e of interest. Good agreement was found between absolute membrane pote ntial data of human peripheral blood lymphocytes by our method and res ults of parallel patch damp measurements.