Biomedical Engineering Reference
In-Depth Information
11 Repeat titration again (i.e. three times in total).
12 The plaques at the greatest dilution for which the cytopathic effect is apparent can be
considered 'plaque pure.'
Notes
c
We expect that the cytopathic effect will be observed by day 3 in the wells infected
with the most concentrated lysate, but that no effect will be observed over the full 8
days in wells infected at the highest dilution. If a cytopathic effect is observed in wells
infected at the highest dilution after 3 days, then collect media and debris as in step 8
and repeat titration but start at higher dilution (e.g. use lysate diluted 10
−
11
as starting
dilution).
PROTOCOL 11.3 Preparation of Crude Lysates of High Titer
Adenovirus Stock from Bulk Cultures of FG293 Cells
Equipment and reagents
•
FG293 cells infected with titrated adenovirus stock (infection of between 5
×
T75 flasks
to 15
×
T175 flasks)
•
1
×
PBS (Sigma)
•
Cell scrapers for T75 or T175 flasks as appropriate (Falcon, Becton Dickinson)
•
50ml conical tubes (Falcon)
•
Bench-top serological centrifuge capable of taking 50ml Falcon tubes
•
PBS++ with 10% glycerol
•
5% sodium deoxycholate solution
•
DNase I (Sigma): dilute to 1mg/ml in 10 m
M
Tris-HCl, pH 7.4; 50 m
M
NaCl; 0.1mg/ml
bovine serum albumin; 1 m
M
dithiothreitol; 50% glycerol (see note d)
1
M
MgCl
2
•
0.45
μ
m low-protein syringe filters (Sartorius)
•
•
20ml syringes.
Method
1 Harvest Ad5 transduced FG293 cells by removing medium and debris from flasks and
pooling in a 50ml Falcon tube.
2Add1
×
PBS to each flask and scrape any remaining cells from flasks, and pool with the
spent medium.
3 Centrifuge the pooled media and cells at 1000
g
for 15 min.
