( ...)Optimizing electroporation parameters
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Other important parameters for optimizing electroporation.
- Field strength
- Pulse length
- Number of field pulses
- Adjustment of the electroporation buffer
- Influence of DNA/RNA
- Influence of temperature
- Influence of the cell density
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. Zimmermann
and G. Neill, editors. pp. 1. CRC Press, Boca Raton, 1996.
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