Biology Reference
In-Depth Information
9. The indicated time points are only a suggestion. Different time
points may also be used. In addition, it is also recommended to
seed three extra noninfected wells as negative controls.
10. HEK-293 cells are poorly attached to the plate surface, espe-
cially after being infected by an adenovirus. All media replace-
ments must be done very gently.
11. Avoid working with confluences higher than 80 % because
highly confluent cells are poorly infected by the adenovirus,
and leads to underestimation.
12. It's recommended to discard the values when the percentage
of positive cells is higher than a 10 %. In higher percentages
probably some of the positive cells are infected by more than
one infectious particle, which will lead to underestimation of
the titer. If most of the time points have percentages of
infection higher than 10 % adjust the dilutions properly from
step 9
and repeat the experiment.
13. Polybrene-mediated enhancing effects on adenovirus infection
are observed only when using Freestyle serum-free medium,
whereas SFMII medium completely blocks the effect of
polybrene. This has also been described for other cationic mol-
ecules such as polyethilenimine (PEI) [
15
].
14. MOI is the number of viral infection units per cell and it
depends on the cell type and the environmental conditions
during infections.
15. The time at which the marker protein is visible at the fluores-
cent microscopy depends on the viral cycle of each vector. For
example, 30 h post-infection for Ad5 or 48 h post-infection for
Ad5/40.
16. HAdV-5/40S produced by 211BS cells are expected to have
both, F5 and F40S proteins (mosaic-chimeric HAdV-5/40S),
whereas HAdV-5/40 produced by 293F cells should only dis-
play F40S on their surface (chimeric HAdV-5/40S). In order
to maximize the amplification of HAdV-5/40S, these vectors
should be grown in 211BS. However, to obtain pure chimeric
(not mosaic) HAdV-5/40S particles, the last step of amplifica-
tion has to be performed in 293F cells.
17. Most viral particles infect the cells during the first 4-6 h post-
infection. After this time, it is important to change the medium
to clear the viral particles that have not entered into the cells
and thus, remove the contaminating chimeric-mosaic particles
used in the infection from the final step.
