The temperature effect during pulse application on cell membrane fluidity and permeabilization.

Cell membrane permeabilization is caused by the application of high intensity electric pulses of short duration. The extent of cell membrane permeabilization depends on electric pulse parameters, characteristics of the electropermeabilization media and properties of cells exposed to electric pulses. In the present study, the temperature effect during pulse application on cell membrane fluidity and permeabilization was determined in two different cell lines: V-79 and B16F-1. While cell membrane fluidity was determined by electron paramagnetic resonance (EPR) method, the cell membrane electropermeabilization was determined by uptake of bleomycin and clonogenic assay. A train of eight rectangular pulses with the amplitude of 500 V/cm, 700 V/cm and 900 V/cm in the duration of 100 micros and with repetition frequency 1 Hz was applied. Immediately after the pulse application, 50 microl droplet of cell suspension was maintained at room temperature in order to allow cell membrane resealing. The cells were then plated for clonogenic assay. The main finding of this study is that the chilling of cell suspension from physiological temperature (of 37 degrees C) to 4 degrees C has significant effect on cell membrane electropermeabilization, leading to lower percent of cell membrane permeabilization. The differences are most pronounced when cells are exposed to electric pulse amplitude of 900 V/cm. At the same time with the decreasing of temperature, the cell membranes become less fluid, with higher order parameters in all three types of domains and higher proportion of domain with highest order parameter. Our results indicate that cell membrane fluidity and domain structure influence the electropermeabilization of cells, however it seems that some other factors may have contributing role.