Biomedical Engineering Reference
In-Depth Information
10. Remove the Effectene-DNA complex by replacing the growth
medium with 20 mL 10A-medium, and incubate for a further
24 h in a 32 °C CO
2
incubator.
11. Plate the MCF10A to be infected the next day: Trypsinize the
MCF10A cells, and determine the cell number using a hemo-
cytometer. Plate six 15 cm diameter dishes with 3 × 10
6
cells
per dish, and allow to grow overnight at 37 °C, 5 % CO
2
.
12. Collect the supernatant from the AMPHO cells, which con-
tains the viral particles. Pool all supernatant together, and fi lter
through 0.45
M cellulose acetate (CA) fi lter units.
13. Aliquot the fi ltered viral supernatant in sterile centrifugation
tubes, and centrifuge at a minimum of 32,000 rcf for 2 h at
4 °C (
see
Note 8
).
14. Carefully remove the supernatant. There will be a very small
transparent pellet on the base of the centrifuge tubes.
15. Resuspend the viral pellets with 90 mL fresh pre-warmed
10A-medium (
see
Note 9
). Add Polybrene to a fi nal concen-
tration of 5
ʼ
g/mL.
16. Remove the medium from the MCF10A cells, and replace it
with the virus-containing medium prepared in Subheading
3.1.1
,
step 14
, evenly distributing the medium over all plates. A total
volume of 15 mL medium suffi ces for each dish.
ʼ
17. Incubate the MCF10A cells at 32 °C for 16-24 h (
see
Note 10
).
18. Trypsinize the infected MCF10A cells, and proceed with soft
agar assay as described in Subheading
3.1.2
.
1. Melt the 3 % agar in a microwave oven at 30 % power and allow
the agar to cool to 41 °C. Warm 41 mL aliquots of 10A-medium
supplemented with 1
3.1.2 Soft Agar Screen
g/mL puromycin to 41 °C. Working
one aliquot at a time, add 9 mL 3 % agar, mix gently by inver-
sion, and apply 25 mL each to two 15 cm dishes. Gently swirl
the dishes to cover the entire surface, without creating air bub-
bles. Set the dishes on a level surface at room temperature, to
solidify (
see
Notes 11
and
12
).
2. To add the top agar layer, heat 3 % agar and cool to 41 °C (
see
Note 13
), and warm 10A-medium supplemented with 1
ʼ
g/
mL puromycin to 37 °C. Dilute the infected MCF10A cells in
tubes of 1.5 × 10
6
cells in 22 mL medium. Working one tube at
a time, add 3 mL 3 % agar, mix gently, and spread 12.5 mL
each to two base agar dishes. Tilt the dishes to evenly spread
the agar-cell layer over the base layer (
see
Note 14
).
3. Allow the top agar layer to solidify completely at room tem-
perature (at least 20 min) before moving the dishes to a 37 °C,
5 % CO
2
incubator (
see
Note 15
).
ʼ
