Flow cytometric estimation of transmembrane potential of macrophages—A comparison with microelectrode measurements

Abstract

Potential‐dependent accumulation of the lipophilic cationic dye 3,3′ dihexyloxacarbocyanine (DiOC₆(3)) in macrophages has been investigated. Resulting fluorescence of cells was measured by flow cytometry. Alterations of membrane potential of macrophages were induced by ionophore treatment (valinomycin and gramicidin) in a dose‐dependent (10⁻⁵ M‐10⁷ M) and time‐dependent (0 min‐45 min) manner. Resulting changes in relative fluorescence intensity were compared with changes of transmembrane potential measured by intracellular recordings obtained by applying glass microelectrodes. The comparative studies offer the possibility to calibrate the flow cytometric estimate of membrane potential of suspended cells. Equilibration of dye partition between cells and surrounding medium is strictly potential‐dependent at dye concentrations between 5 × 10⁻⁸ M and 10⁻⁷ M and within an incubation interval from 10 min up to 30 min after addition of dye. Conclusions are drawn concerning the field of application of the optical method. Dynamics of electrical processes following ionophore treatment are discussed in terms of molecular mechanisms of altered ionic transport.