Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells

Abstract

The effects of ionic composition and conductivity of the medium on electropermeabilization of the plasma membrane of mammalian cells were studied. Temporal and spatial uptake of propidium iodide (PI) into field-treated cells was measured by means of flow cytometry, spectrofluorimetry and confocal laser scanning microscopy. Murine myeloma cells were electropulsed in iso-osmolar solutions. These contained 10–100 μg ml −1 PI at different conductivities (0.8–14 mS cm −1) and ionic strengths, adjusted by varying concentrations of K +, Na +, Cl − and SO 4 2−. Field-induced incorporation of PI into reversibly permeabilized cells was (almost) independent of ionic composition and strength (at a fixed medium conductivity), but increased dramatically with decreasing medium conductivity at a fixed field strength. The time-course of PI uptake (which apparently reflected the resealing process of the membrane) could be fitted by a single-exponential curve (relaxation time about 2 min in the absence of Ca 2+) and was independent of medium conductivity and composition. These and other data suggested that the expansion of the ‘electroleaks’ during the breakdown process is field-controlled and depends, therefore, on the (conductivity-dependent) discharging process of the permeabilized membrane. The threshold field strength for dye uptake increased with increasing K + concentration (about 0.6 kV cm −1 in K +-free, NaCl-containing medium and about 0.9 kV cm −1 in 30 mM KCl-containing medium). Also, the spatial uptake pattern of PI shifted from an asymmetric permeation through the cell hemisphere facing the anode to a more symmetric uptake through both hemispheres. These results suggested that the generated potential is superimposed on the (K +-dependent) resting membrane potential. Taking this into account, the breakdown voltage of the membrane was estimated to be about 1 V.