Biomedical Engineering Reference
In-Depth Information
PROTOCOL 11.7 Transfection
Equipment and reagents
Cells to be transfected
p
•
Complete culture medium
q
•
Culture medium without supplements
r
•
Culture medium with double concentration of supplements
s
•
Transgene detection assay.
t
•
Method
Day 1
1 Seed cells into 24-well plates at 2
×
10
5
per well in complete culture medium without
antibiotics.
u
2 Incubate overnight at 37
◦
C, 95% air/5% CO
2
.
Day 2
1 Prepare complexes as described above in Protocols 11.5 and 11.6.
2 Remove medium and add 1ml per well culture medium (without supplements) to wash
cells.
3 Remove culture medium and add 0.5ml vector-DNA complexes per well.
4 Incubate at 37
◦
C in 95% air/5% CO
2
for 4 h.
5 Add 0.5ml per well culture medium containing twice the concentration of supplements
and place cells back into the incubator.
v
Day 3
1 Harvest cells for transgene expression, according to detection assay.
Notes
p
When commencing transfection experiments, choose a relevant target cell line that is easy
to grow. Cell lines and stocks of DNA and vectors should be prepared and cultured under
mycoplasma-free conditions. The presence of mycoplasma can reduce transfection levels.
q
Culture medium specific to the target cells containing all the necessary growth supplements,
including serum, l-glutamine, and so on. Concentrations of supplements are cell-type dependent.
r
The same culture medium as in note q, but containing no added supplements.
s
Complete culture medium, as in note q, containing double the concentration of added
supplements.
t
Assay to measure the level of transgene expression. Reporter gene assays are discussed in
Section 11.2.8.
u
Cells need to be 80-90% confluent on the day of the transfection.
v
If harvesting at any time point
>
48 h, the medium must be replaced at 24 h with complete
culture medium.
