Eppendorf Multiporator - Electroporation - Manual (Page 9)

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To ensure that maximum transfection rates are achieved, the electroporation parameters should be optimized for each
new cell line. This section contains guidelines for determining the ideal parameters as simply and as quickly as possible.
The field strength (V/cm) of the electrical pulse used is an essential factor in determining the survival rate as well as the
transfection rate of the cells used.
If the field strength of the pulse exceeds a characteristic value (= critical external field strength), reversible permeation
occurs in the cell membrane. This so-called permeation voltage is heavily dependent on the cell diameter and the
temperature at which electroporation takes place. The diagrams in Fig. 2 show the permeation voltage that has to be set
in relation to the cell diameter and the temperature at which the electroporation is performed. The diameter of the cell is
determined after the cells have been incubated in electroporation buffer for 10 – 15 minutes (see Sec. 3.4 + 7.1).
In addition, the gap width of the cuvettes must be taken into account when the minimum pulse voltage is determined.
If the gap width is doubled, the pulse voltage must also be doubled in order to obtain the same field strength. A general
rule when determining the ideal field strength is that small cells require a higher field strength in order to achieve
membrane permeation. The pulse voltages in Fig. 2 and Table 2 (page 16) are the minimum values at which the
membrane can be permeated. However, depending on the cell type used, optimal transfection efficiency is often only
achieved at significantly higher voltages. To determine the optimal pulse voltage, it is advisable to carry out a series of
experiments in which the minimum value, twice the value and then three times the value shown in Table 2 (page 16) are
used for suspension cells, and up to five times the value for adherent cells. Cells which do not assume a rounded form in
the electroporation buffer often require even higher pulse voltages before optimal transfection can occur.
Please note that increasing the pulse voltage can increase the transfection rate but, at the same time, can also increase
the cell mortality rate.
Fig. 2: Minimum pulse voltage at which the cell membrane is permeated
3.1 Optimizing the field strength
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
0 20 40 60 80 100
4 °C
RT
Cuvette: 2 mm gap width
400 µl volume
V
oltage [V]
Cell diameter [µm]
0
4 0 0
8 0 0
1 2 0 0
1 6 0 0
2 0 0 0
2 4 0 0
2 8 0 0
0 20 40 60 80 100
4 °C
RT
Cuvette: 4 mm gap width
800 µl volume
V
oltage [V]
Cell diameter [µm]
3 Optimizing the parameters
3 Optimizing the parameters
Multipor_Appli_E_poration_en.fm Seite 33 Montag, 30. Januar 2006 2:17 14