Biology Reference
In-Depth Information
7. On the next day take an aliquot of the suspension, microscopi-
cally examine and count the cells. Add 0.5 L of fresh medium
supplemented as described in
step 4
.
8. On the next day take an aliquot of the suspension, microscopi-
cally examine and count the cells. Add 1.0 L of fresh medium
supplemented as described in
step 4
.
9. On the next day take an aliquot of the cell suspension
and count the cells. The cell density should be between 2 and
4E5 cells/mL at the time of coinfection with helper virus
and HC-Ad vector. In addition, the cells should not have
formed clumps.
10. When the cells reach the required density, harvest the cells by
centrifugation at 400 ×
g
for 10 min.
11. Resuspend the cells in 100 mL of the spent medium and trans-
fer them to a fresh 250-mL spinner fl ask.
12. Infect cells with 200−300 physical particles of a purifi ed and
characterized HC-Ad vector preparation and coinfect with
15−20 MOI helper virus.
13. Incubate the cells for 2 h at 37 °C while stirring at 60 rpm.
14. Transfer the cells including the medium to a fresh 3 L spinner
fl ask and fi ll up to 3 L with fresh medium.
15. Incubate for 48 h while stirring at 60 rpm.
16. Harvest the cells by centrifugation at 400 ×
g
and resuspend in
4 mL PBS per 2E8 cells.
17. Continue with the purifi cation protocol described in
Subheading
3.4
(
see
Notes 21
and
22
).
1. HC-Ad vectors can be stored long-term (>4 years) at −80 °C
when supplemented with 10 % glycerol as cryoprotectant.
2. Avoid repeated freezing and thawing of HC-Ad vector prepa-
rations by preparing appropriately sized aliquots before the
fi rst freezing.
3. Avoid prolonged storage at room temperature.
4. Avoid storage in glass containers. Depending on the nature of
the glass the vector may irreversibly adhere to the glass
surface.
5. Avoid vortexing or other means of vigorous mixing.
6. When shipping on dry ice prevent CO
2
from entering the
packaging of the vector. It is best to place the vector tubes
wrapped into parafi lm into several layers of CO
2
-tight plastic
bags, which are vacuum sealed.
3.8 Handling,
Storage, and Shipping
