Evaluation of cell membrane electropermeabilization by means of a nonpermeant cytotoxic agent

For the evaluation of cell membrane electropermeabilization, cells are usua lly exposed to electric pulses in the presence of propidium iodide, a fluor escent dye activated by binding to cellular DNA. The fraction of permeabili zed cells is then determined using a flow cytometer. This widely establishe d method has several drawbacks: (i) an arbitrary choice of minimum fluoresc ence intensity for characterization of permeabilized cells; (ii) the inabil ity to detect cells disintegrated because of intense electropermeabilizatio n; and (iii) false detection of cellular ghosts devoid of fluorescence beca use of leakage of DNA caused by electropermeabilization. Here, we present a simple and inexpensive method that eliminates these drawbacks. The method is based on the use of a cytotoxic agent that cannot permeate through an in tact plasma membrane and thus leads to selective death of the electropermea bilized cells. The amount of nonpermeabilized cells is then determined by a suitable viability test. Bleomycin at a 5-nM concentration causes no stati stically significant effect on cell survival in the absence of electric pul ses, yet this concentration is sufficient for lethal toxicity in electroper meabilized cells. The amount of cells surviving the exposure relative to th e control gives a reliable value of the fraction of nonpermeabilized cells.