ELECTROPERMEABILIZATION AND FLUORESCENT TRACER EXCHANGE - THE ROLE OFWHOLE-CELL CAPACITANCE

Transmembrane crossing of charged fluorescent tracers such as propidiu m iodide (PI) and carboxy-fluorescein (CF) can be used to quantitate m embrane permeabilization. Murine myeloma Sp2/0-Ag14 cells were loaded with CF (0.1 fmol/cell) before electropulsation (0.5-3.0 kV/cm, 40 mu s) in medium containing 25-50 mu g/ml PI at 21-23 degrees C. Cytograms of PI vs, CF fluorescence showed three readily distinguishable subpop ulations: 1) intact living cells with CF but without PI (these form > 95% of the prepulsed population), 2) transiently electropermeabilized but resealed cells showing both CF and low-level PI fluorescence, and 3) permanently permeabilized cells without CF but with very high PI fl uorescence. Despite the ready influx of PI, the efflux of CF from tran siently permeabilized cells was negligible and was insensitive to puls e parameters; however, electrically killed cells (subpopulation 3) los t all CF fluorescence and probably lost their cytoplasm, This differen ce in transmembrane passage of the dyes is best explained by binding o f intracellular CF to macromolecoles (and/or organelles), In isotonic ''pulse medium,'' the membranes resealed after electropulsing with a t ime constant (tau(R)) of about 2 min, La 150 mOsm medium, resealing wa s faster (typically tau(R) similar to 0.5 min). The population distrib ution of PI uptake [coefficient of variation (CV) > 40%] was very broa d and could not be accounted for by the radius dependence of pulse-ind uced voltage (CVradius similar to 10%), The variability in PI uptake c ould be explained if the electrical energy of the charged membrane, wh ich depends on the whole-cell capacitance (C-c), was taken into accoun t, Evaluation of the C-c values with single-cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation. (C) 1995 Wiley-Liss, Inc.