Biology Reference
In-Depth Information
Fig.
2
Amplification strategy of chimeric HAdV-5/40S vectors. The first amplification an intermediate steps are
performed in 211BS cells. Last step is carried out in 293F cells to obtain pure chimeric HAd5V-/40S vectors
1. Seed 10
6
211BS cells/mL in one 125 mL shake flask (25 mL
working volume) in Infection Medium. Keep the culture in
suspension by agitation in an orbital shaker at a speed of
110 rpm, 37 °C and 5 % CO
2
.
2. Add polybrene to a final concentration of 9 μg/mL (
see
Note 13
).
3. Infect cells with the vector pre-stock at an MOI of 1 (
see
Note 14
).
4. Four hours post-infection, supplement the cell culture with
0.5 % FBS.
5. Optional step: if vector expresses a fluorescent marker protein,
such as GFP, the infection efficiency can be estimated by fluo-
rescent microscopy at 30 h post-infection (
see
Note 15
).
6. Harvest cell cultures at 56 h post-infection and store at −80 °C.
7. Lyse cells by three freeze/thaw cycles in order to release the
virus from cells.
8. Centrifuge at 1,620 ×
g
for 5 min to remove cell debris.
9. Store at −80 °C.
