Electroporation: the population distribution of macromolecular uptake and shape changes in red blood cells following a single 50 μs square wave pulse

We describe a method of using flow cytometry for determining the distribution of electroporation effects within a statistically significant cell population. Here we illustrate basic aspects of the method by investigating the electroporation of red blood cells, which have been widely used by others in previous investigations of electroporation, including studies of reversible electrical breakdown, and molecular uptake or release associated with a transient high permeability state. We make two measurements on each cell in a population of 10,000 or more cells: (1) light scatter to indicate changes in cell morphology, and (2) fluorescence to determine the uptake of a fluorescence-labeled macromolecule (FITC-dextran; 70,000 dalton). Computer processing of the single cell data allows construction of statistical distributions which reveal how electroporation occurs within a large cell population. Using this method we find that after a single 50 μs square pulse of optimal magnitude about 30% have formed spherical ghosts because of electroporation, and that two distinct ghost subpopulations occur. One subpopulation (about 10% of analyzed cells) has negligible uptake, while the second subpopulation (about 20%) consists of ghosts which have taken up significant amounts of the test macromolecule. Two interesting findings are the high frequency-of-occurrence of electroporation due to a single, optimal pulse, and the implication, because of the two distinct ghost subpopulations, that there is a significant variation in pore sizes.