Using the Neon
™
Transfection System,
Continued
Preparing
Adherent Cells
1.
Cultivate the required number of cells (see below).
2.
One–two days prior to electroporation, transfer the cells into flask with fresh
growth medium such that the cells are 70–90% confluent on the day of the
experiment.
5 × 10
4
–2 × 10
5
cells per each 10 μL Neon
™
Tip for most optimized protocols.
5 × 10
5
–2 × 10
6
cells per each 100 μL Neon
™
Tip for most optimized protocols.
3.
Pre-warm an aliquot of culture medium containing serum, PBS (without Ca
2+
and Mg
2+
), and Trypsin/EDTA solution to 37ºC.
4.
Aspirate the media from cells and rinse the cells using PBS (without Ca
2+
and
Mg
2+
).
5.
Trypsinize the cells using Trypsin/EDTA or TrypLE Express (Cat. no. 12563).
6.
After neutralization, harvest the cells in growth medium with serum
(~0.75 mL for 10 μL Neon
™
Tip or 7.5 mL for 100 μL Neon
™
Tip).
7.
Take an aliquot of trypsinized cell suspension and count cells to determine
the cell density.
8.
Transfer the cells to a 1.5 mL microcentrifuge tube or a 15 mL conical tube
and centrifuge the cells at 100–400 × g for 5 minutes at room temperature.
9.
Wash cells with PBS (without Ca
2+
and Mg
2+
) by centrifugation at 100–400 × g
for 5 minutes at room temperature.
10.
Aspirate the PBS and resuspend the cell pellet in Resuspension Buffer R at a
final density of 1.0 × 10
7
cells/mL. Gently pipette the cells to obtain a single
cell suspension.
Note:
Avoid storing the cell suspension for more than 15–30 minutes at room
temperature, which reduces cell viability and transfection efficiency. The resuspension
cell density may be adjusted to accommodate the recommended cell numbers for the
electroporation protocol (page 18) or optimization protocols (pages 24–29).
11.
Prepare 24-well plates by filling the wells with 0.5 mL of culture medium
containing serum and supplements
without antibiotics
and pre-incubate
plates in a humidified 37°C/5% CO
2
incubator. If you are using other plate
format, see page 18 for plating medium volume recommendations.
Continued on next page
16
