Author/Editor		Kotnik, Tadej; Maček-Lebar, Alenka; Miklavčič, Damijan; Mir, Lluis M
Title		Evaluation of cell membrane electropermeabilization by means of a nonpermeant cytotoxic agent
Type		članek
Source		In: Jarm T, Kotnik T, Miklavčič D, editors. Proceedings including program of the 3rd international conference on bioelectromagnetism and 1st Slovenian-Croatian meeting on biomedical engineering in connection with COST 244bis 11th workshop on biomedical applications of electromagnetic fields and management commitee meeting; 2000 oct 8-12; Bled. Ljubljana: Faculty of electrical engineering,
Publication year		2000
Volume		str. 9-10
Language		eng
Abstract		Cell membrane electropermeabilization is usually evaluated by exposing the cells to electric pulses in presence of propidium iodide, a fluorescent dye activated by binding to cellular DNA. The fraction of permeabilized cells is then determined using a flow cytometer. This widely established method has several drawbacks: (i) an arbitrary choice of minimum fluorescence intensity for characterization of permeabilized cells; (ii) the inability to detect cells disintegrated due to intense electropermeabilization; and (iii) false detection of cellular ghosts devoid of fluorescence due to leakage of DNA caused by electropermeabilization. In this paper, 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 intact plasma membrane, and thus leads to selective death of the electropermeabilized cells. The amount of nonpermeabilized cells is then determined by a suitable viability test. Bleomycin at 5 nM concentration causes no statistically significant effect on cell survival in absence of electric pulses, while this concentration is sufficient for lethal toxicity in electropermeabilized cells. The amount of cells surviving the exposure relative to the control gives a reliable value of the fraction of nonpermeabilized cells.
Descriptors		CELL MEMBRANE PERMEABILITY BLEOMYCIN ELECTROPORATION FLOW CYTOMETRY PROPIDIUM CELL LINE, TRANSFORMED FIBROBLASTS