Multiple-pulse electroporation: Uptake of a macromolecule by individual cells of Saccharomyces cerevisiae

Abstract

The “green” fluorescence (GF) of individual yeast cells ( Saccharomyces cerevisiae) was measured by flow cytometry to determine the amount of uptake of a macromolecule (FITC-dextran) due to the application of one or more square 50 μs electric field pulses. A second molecule, propidium iodide, was added 25 min after pulsing. The number of pulses N pulse was varied by factors of two, up to N pulse = 16, with individual pulses separated by 5 s. At the lowest electric field, E e = 1.5 kV cm −1 increasing N pulse up to 16 did not result in enhanced uptake of either molecule. At the next higher field (3 kV cm −1) there was a dramatic increase in both the percentage of cells with GF, and also the mean GF per cell, when N pulse was increased from 1 to 2, and then from 2 to 4. For this pulse size a further increase in pulse number from 4 to 8 resulted in a decrease in the percentage of participating cells (mean percentage of cells with GF), but an increase in the average GF. At the optimal single pulse electric field strength (5 kV cm −1), increasing the number of pulses did not increase the percentage of cells with GF, although there was some increase in the percentage of cells with red fluorescence (RF); increasing N pulse at the largest field (7 kV cm −1) did not result in increased uptake of the dextran. However, an increase in the number of cells which take up propidium iodide was observed, which appears to correlate with increased cell death. The number of viable cells remaining after pulsing was estimated by propidium iodide uptake. This rapid estimate was compared with traditional viable plating at 5 kV cm −1 and found to give a reasonable correlation for this microorganism under the conditions of this study.