Using the Neon
™
Transfection System,
Continued
Preparing
Suspension Cells
1.
Cultivate the required number of cells (see below).
2.
One to 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. For most cell lines, the cell density is ~1–3× 10
6
cells/T-
25 flask.
1–5 × 10
5
cells per each 10 μL Neon
™
Tip for most optimized protocols.
1–5 × 10
6
cells per each 100 μL Neon
™
Tip for most optimized protocols.
3.
Pre-warm an aliquot (500 μL per sample for 10 μL Neon
™
Tips or 5 mL for
100 μL Neon
™
Tips) of culture medium containing serum. Also prepare an
appropriate volume of PBS (without Ca
2+
and Mg
2+
).
4.
Take an aliquot of cell culture and count the cells to determine the cell
density.
5.
Transfer the cells to a microcentrifuge tube or 15 mL conical tube and pellet
the cells by centrifugation at 100–400 × g for 5 minutes at room
temperature.
6.
Wash the cells with PBS (without Ca
2+
and Mg
2+
) and pellet the cells by
centrifugation at 100–400 × g for 5 minutes at room temperature.
7.
Aspirate the PBS and resuspend the cell pellet in Resuspension Buffer R or
Resuspension Buffer T at a final density of 2.0 × 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 maybe adjusted to accommodate the recommended cell
numbers for the electroporation protocol (page 18) or optimization protocols (pages
24–29).
8.
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
17
