Fluorescent indicators of membrane permeabilization due to electroporation

Abstract

The plasma membrane represents a barrier between a cell and its surroundings. The membrane's selective transport enables the cell to maintain solute concentrations different from the outside medium. However, in the process of electroporation, the function of plasma membrane barrier and transport selectivity is at least partially lost and the molecules that are impermeant to intact membrane can enter into the cell's cytoplasm. The uptake of such molecules can be indicative for plasma membrane permeabilization. Most commonly used indicators for this purpose are fluorescent molecules: their cellular uptake can be detected by fluorescence microscopy as well as by methods suitable to analyze bulk cells, such as spectrofluorometers, plate readers, and flow cytometers. The use of fluorescent indicators is a simple, noninvasive, highly sensitive, and safe method to monitor the extent of membrane permeabilization, allowing real-time dynamic imaging or evaluating response of an average response to electric pulse exposure, depending on the mode of detection used. There are many fluorescent indicators that are used in electroporation experiments. Among them, the most widely used fluorophore is propidium iodide: because it significantly increases its fluorescence by binding to nucleic acids, it enables microscopic observation of rapid transport across permeabilized membrane without any washing. In addition to nucleic acid binding dyes (e.g., PI, YO-PRO®-1 Iodide), there are also other fluorescent indicators of membrane permeabilization: nonbinding, low molecular weight (such as lucifer yellow), and large indicators (such as dextrans and quantum dots) available on the market. With the use of different fluorescent indicators and detection methods, scientists can gain insight on different perspectives of the electroporation phenomenon (e.g., asymmetric uptake of molecules, the effects of pulse parameters on membrane permeabilization, the size of pores, and response variability in cells).