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Optimizing electroporation parameters

Other important parameters for optimizing electroporation.
Influence of temperature

The temperature has a direct effect on the permeation voltage of the cell membrane (see field strength) as well as on the regeneration of the membrane following electroporation.

a) Influence of the temperature on the permeation voltage of the cell membrane:
Since the permeation voltage at 4C is twice that at room temperature (1), it is essential to take the temperature into account when determining the optimal field strength of the pulse. During electroporation at 4C, the necessary field strength of the pulse is nearly twice as high as those values for room temperature. However, mammalian cells are usually electroporated at room temperature.

b) Influence of the temperature on the regeneration of the cell membrane:
Incubating cells following electroporation at low temperatures (e.g. 4C) slows down the healing process of the cell membrane. In the case of eukaryotic cells, the resealing of the membrane pores can take half an hour or longer under these conditions. With certain cell types, this can lead to an increase in the amount of transfection material absorbed. However, some cells are extremely sensitive to low temperatures, particularly when permeated, and can suffer from irreversible damage after short incubation times in a cold environment. In those cases where electroporation at 4C leads to higher transfection rates, the cells' chances of survival can be boosted if they are resuspended in electroporation buffer at 37C or room temperature, cooled down to 4C and then transferred into precooled cuvettes. Following electroporation, the cells are cooled on ice for a maximum of two minutes and then heated to 37C in a water bath. Electroporation at higher temperatures (e.g. 25C) causes the permeated membrane areas to seal up more rapidly, which accelerates membrane regeneration and thus increases the cell survival rate. However, the transfection rate may be lower than that obtained when electroporation is carried out at low temperatures.

c) Influence of the temperature on the conductivity of the buffer:
The temperature has a profound effect on the conductivity of the electroporation buffer. Increasing the temperature causes the conductivity of a solution to increase as well, which may lead to lower transfection rates. For this reason, it is advisable not to work at temperatures in excess of 33C

(1) Zimmermann U. (1996) Effect of high intensity electric field pulses on eukaryotic cell membranes. In: Electromanipulation of Cells. U. Zi mmermann and G. Neill, editors. pp. 1. CRC Press, Boca Raton, 1996.



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