Biology Reference
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5. Harvest the samples at the indicated times. Collect both super-
natant and cells in a tube and store it at −80 °C. Repeat for all
the sampling times.
6. Freeze/thaw three times.
7. Thaw the samples and centrifuge for 5 min at 4,500 ×
g
. Transfer
the supernatant into a new tube and discard the pellet.
8. Plate new HEK-293 cells in a 96-well plate at a 70 % of conflu-
ence to titrate the samples (
see
Note 11
).
9. From each sample in
step 7
save volumes of 10, 1, 0.1, or
0.01 μL and dilute to a total volume of 100 μL of infection
medium.
10. Forty-eight hours after the infection count the infected cells
by using the reporter gene or the hexon-antibody immuno-
cytochemistry-based protocol. Calculate the IU/mL per
sample, as described in Subheading
3.3.1
. The time point with
the highest value is the optimal time for harvesting the vector.
As observed in Fig.
1
, 56 h is the recommended harvesting
time for the chimeric HAdV5-40s vector (
see
Note 12
).
3.6 Chimeric
Adenoviral Production
from a Pre-stock
1. Plate twenty 150-mm petri dishes with HEK-293 cells at a
confluency of 70 %. Add growing medium up to a final volume
of 18 mL.
2. Twenty-four hours later, replace the growing medium with
12 mL of infection medium.
3. Calculate the amount of virus to infect twenty 150-mm plates
with an MOI of 5, and dilute in 20 mL of infection medium.
Add 1 mL of the mix per plate.
Fig.
1
Replicative cell cycle of the chimeric HAdV-5/40S vector in HEK-293 cells. According to this, the
replicative cycle is between 48 and 56 h post-infection. The values are represented as percentage of the
highest titer obtained
