4. Bilevel electric pulses are applied for electroporation (see
Note 7).
4 Notes
1. PC membrane pore size is an important parameter because it
determines the size of the pore generated in the cell membrane
and thus influences cell viability and cargo delivery. Smaller
pores result in higher viability and dosage control yet limit
cargo size. PC membrane pore size also affects the membrane
channel resistance and therefore the voltage delivered to the
cell membrane. The most effective pore size choices are 50 nm
and 200 nm.
2. The function generator is used to produce the electric field for
electroporation and therefore must be able to operate at the
chosen frequency and voltages. The Bio-Rad Gene Pulser II is a
commercialized electroporation system which can also be used
for this purpose.
3. Oxygen plasma cleaning surfaces makes them more hydrophilic, allowing them to bond to other hydrophilic surfaces.
Oxygen plasma cleaning also reduces the conductivity of ITO
coatings, so exposure of the glass slides should be minimized
[13]. However, this effect is negligible for this experiment due
to the relatively low resistance of the ITO compared to the
resistances of the PC membrane and the cell membrane.
4. These settings are recommended for culturing HeLa or
HT1080 cells but may vary depending on the cell line used.
5. Concentrations of 0.01–1 μg μL
À1 have been used for this
experiment. Concentrations of 0.01 or 0.02 μg μL
À1 are
recommended because values closer to 1 μg μL
À1 yielded a
lower transfection efficiency (Kang et al. [9]).
6. The resistance between the two electrodes can vary significantly
based on the PC membrane pore size.
7. The parameters for the square wave electric pulses depend on
the PC membrane selected (see Note 1). Generally, pulses are
most effective when they consist of a short high voltage pulse
alternated with a long low voltage pulse [14], but this was not
performed with this device. The high voltage pulse must
exceed 0.2–1 V at the membrane for transfection to occur
[15]. For HeLa cells on a PC membrane with pores that are
2 μm in diameter and 24 μm in length, high voltage pulses of
10–20 V have been used and 7.7 V is the theoretical minimum
[9]. Experiments were performed with 1–3 pulses that were
0.25–0.75 s in duration and at a frequency of 200 Hz. Further
experiments were conducted to determine parameters for
HT1080 cells and postmitotic neurons.
96
Justin Brooks et al.
Note 7).
4 Notes
1. PC membrane pore size is an important parameter because it
determines the size of the pore generated in the cell membrane
and thus influences cell viability and cargo delivery. Smaller
pores result in higher viability and dosage control yet limit
cargo size. PC membrane pore size also affects the membrane
channel resistance and therefore the voltage delivered to the
cell membrane. The most effective pore size choices are 50 nm
and 200 nm.
2. The function generator is used to produce the electric field for
electroporation and therefore must be able to operate at the
chosen frequency and voltages. The Bio-Rad Gene Pulser II is a
commercialized electroporation system which can also be used
for this purpose.
3. Oxygen plasma cleaning surfaces makes them more hydrophilic, allowing them to bond to other hydrophilic surfaces.
Oxygen plasma cleaning also reduces the conductivity of ITO
coatings, so exposure of the glass slides should be minimized
[13]. However, this effect is negligible for this experiment due
to the relatively low resistance of the ITO compared to the
resistances of the PC membrane and the cell membrane.
4. These settings are recommended for culturing HeLa or
HT1080 cells but may vary depending on the cell line used.
5. Concentrations of 0.01–1 μg μL
À1 have been used for this
experiment. Concentrations of 0.01 or 0.02 μg μL
À1 are
recommended because values closer to 1 μg μL
À1 yielded a
lower transfection efficiency (Kang et al. [9]).
6. The resistance between the two electrodes can vary significantly
based on the PC membrane pore size.
7. The parameters for the square wave electric pulses depend on
the PC membrane selected (see Note 1). Generally, pulses are
most effective when they consist of a short high voltage pulse
alternated with a long low voltage pulse [14], but this was not
performed with this device. The high voltage pulse must
exceed 0.2–1 V at the membrane for transfection to occur
[15]. For HeLa cells on a PC membrane with pores that are
2 μm in diameter and 24 μm in length, high voltage pulses of
10–20 V have been used and 7.7 V is the theoretical minimum
[9]. Experiments were performed with 1–3 pulses that were
0.25–0.75 s in duration and at a frequency of 200 Hz. Further
experiments were conducted to determine parameters for
HT1080 cells and postmitotic neurons.
96
Justin Brooks et al.