Biomedical Engineering Reference
In-Depth Information
Phase microscope
•
80
◦
C freezer.
•
Method
1 Day 0: seed FG293 cells onto 96-well plate from growing stocks, to achieve 50-60%
confluency the following day (approximately 2
×
10
4
cells per well).
2 Day 1: prepare serial dilutions of crude adenovirus lysate (see Table 11.2 in cell culture
medium I.
Table 11.2
Dilution curve for titration of recombinant adenovirus.
Row number
Final virus dilution
Virus volume (
μ
l) Media volume (
μ
l)
10
−
2
-
50 stock
4950
A
10
−
4
50 of 10
−
2
dil
4950
10
−
6
50 of 10
−
4
dil
B
4950
10
−
7
500 of 10
−
6
dil
C
4500
10
−
8
500 of 10
−
7
dil
D
4500
10
−
9
500 of 10
−
8
dil
E
4500
10
−
10
500 of 10
−
9
dil
F
4500
10
−
11
500 of 10
−
10
dil
G
4500
H
0
0
5000
3 Remove the medium from all wells and replace with diluted virus stock (200
μ
l/well).
Use medium alone for control wells.
4 Incubate the plate overnight at 37
◦
C.
5 Day 2: remove medium and replace with 200
μ
l fresh cell culture medium I.
6 Replace medium every 2-3 days for 8 days.
c
Before removing the medium, observe each
well; once the cytopathic effect is apparent in a well, mark it and stop changing the
medium in that well.
7 Day 7: prepare a fresh 96-well plate of FG293 cells for a further round of plaque
purification.
8 Day 8: collect the cells and medium from three wells at the highest dilution (lowest
starting concentration of lysate) for which the cytopathic effect is apparent.
9 Store plaques in separate sterile eppendorfs. These plaques will have arisen from a single
adenovirus virion. Freeze two and extract the third by three freeze-thaw cycles.
10 Repeat the plaque purification assay with the plate prepared on day 7 using the plaque
isolated and lysed by freeze-thaw in step 9 above.
