Biomedical Engineering Reference
In-Depth Information
7 When cytopathic effect is observed (i.e. large 'holes' in monolayer with lots of
rounded-up cells, this can take up to 3 weeks
b
and looks different to overgrown
monolayer on control flask; see Figure 11.2). Do not replace medium and leave for a
further 48 h before harvesting.
(a)
(b)
(c)
Figure 11.2
Monitoring of infection of 293 cells with Ad5 vectors. Sub-confluent 293
cells were infected with Ad vectors and observed after 36 h. (a) Non-infected 293 cells.
(b) Infection of 293 with low titer Ad5. (c) Infection of 293 with high titer Ad5. Note
the 'bunch of grapes' morphology of the infected cells as the virus's lytic life cycle has a
cytopathic effect on the mammalian cells.
8 To harvest, remove medium from transfected flasks and pool in 50ml Falcon tube, add
5ml1
×
PBS
++
to each flask and scrape remaining cells from flasks.
9 Pool with the spent medium and centrifuge at 1000
g
for 15min.
10 Discard the supernatant and resuspend the cell pellet in 150-200
μ
lPBS
++
supplemented with 10% glycerol per T25 harvested flask.
11 Freeze at
−
70
◦
C and thaw at 37
◦
C three times prior to titration.
12 Aliquot and store at
−
70
◦
C.
Notes
a
Sterilize by filtration or autoclaving; store at 4
◦
C.
b
If cytopathic effect is observed rapidly (death of all cells within a few days) then collect the
supernatant and debris. Follow steps 9-11 above and then reinfect new T25 flasks of FG293
cells, using increasing dilutions of the crude lysate prepared in step 11 (e.g. 10
μ
l lysate; 5
μ
l
lysate; 1
μ
l lysate).
PROTOCOL 11.2 Plaque Purification by End-Point Dilution
and Titration of Crude Virus Stock
Equipment and reagents
FG293 human embryonic kidney cells (Microbix Systems Inc, http://microbix.com)
•
Cell culture media I: minimal essential medium, glutamine, 10% FCS (all Sigma)
•
96-well tissue culture plates
•
