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Optimizing electroporation parameters

Other important parameters for optimizing electroporation.
Field Strength 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 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
5 530 1100 1100
10 270 540 540 1100
15 180 360 360 710
20 130 260 260 530
25 110 220 220 430
30 90 180 180 360
35 80 160 160 310
40 70 140 140 270
45 60 120 120 240
50 50 100 100 210
60 40 80 80 160
80 30 60 60 120
Table 1. Minimum pulse voltages at which the cell membranes may be permeated, in relation to the diameter of the cell after a 1015 min incubation in the electroporation buffer, the electroporation temperature and the gap width of the cuvette. Note: Depending on the cell line, the optimal pulse voltage for the electroporation experiment may be between two and five times higher. Figure 1. Minimum pulse voltage at which the cell membrane is permeated. The minimum pulse voltage is dependent on the cell diameter following incubation in electroporation buffer, as well as on the temperature, and on the gap width of the cuvettes. The values shown can be used to determine the ideal pulse voltage to be set on the device.

Figure 2. Cells swell up under hypoosmolar conditions. In this state, electroporation can be performed under milder pulse conditions.

Example:

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



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