If the field strength of the pulse exceeds a specific threshhold (= critical external field strength), reversible permeation occurs in the cell membrane. This so-called permeation voltage is heavily dependent on the temperature at which electroporation takes place. The diagrams in Fig. 1 show the minimum values of the pulse voltage which have 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 1015 minutes (see adjustment of electroporation buffer). 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.1 and the corresponding Table 1 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 1 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 (see Fig. 2) 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.
|Cell diameter, in m||Voltage 2 mm room temp.||Voltage 4 mm room temp. < /b>||Voltage 2 mm 4C||Voltage 4 mm 4C|
Cell type: Suspension cells
Cell diameter in electroporation medium: ~ 20 m
Gap width of cuvette: 2 mm
Temperature in the cuvette: Room temperature
Minimum pulse voltage according to diagrams (Fig. 1): ~ 130 V
Series of experiments for optimizing pulse voltage: 130 V / 260 V / 390 V