2. Verify that the virus titer is known, which is needed to calculate
the volume of virus required to produce the desired MOI.
General formulae:
Virus
IU
well
À Á ¼ seeding density
cells
well
À Á Â MOI
IU
cell
À Á
Vol:of Virus
μl
well
¼
Virus IU=well
ð
Þ
Viral titer IU=μl
ð
Þ
Example: Calculating the amount of virus required in a 24-well
plate.
I. Amount of virus needed for transduction:
Seeding density ¼ 5 Â 10
4 cells
well
À Á
; MOI ¼ 200
Virus
IU
well
À Á ¼ 5 Â 10
4 cells
well
À Á Â 200
IU
cell
À Á ¼ 1 Â 10
7 IU
well
À Á
II. Transforming IU into volume of virus needed per well:
Viral titer ¼ 1 Â 10
10 IU
ml
À Á ¼ 1 Â 10
7 IU
μl
;
Virusð
IU
well Þ ¼ 1 Â 10
7
ð
IU
well Þ
Therefore,
Vol:of Virusð
μl
well Þ ¼
1Â10
7 ð
IU
well Þ
1Â10
7 ð
IU
μl Þ
¼ 1ð
μl
well Þ
As a result, 1 μl of the viral stock is needed to transduce 5 Â 10
4
cells with a MOI of 200.
3.3 PEGylating
Adenovirus
The following procedure is designed to produce 10
7 CPP-PEG-Ad
particles, which in our case is suitable for transducing one well of a
24-well plate. The reaction can be scaled to transduce a greater
number of wells or a larger plate.
1. Dilute 1 μl of virus from the purified viral stock into 99 μl of
50 mM HEPES buffer (pH 8.0) to reach 100 μl. Keep the virus
on ice until used.
2. Prepare 1 ml of stock solution of 1 mg/ml MAL-PEG-NHS
(5 kDa) in 50 mM HEPES buffer (pH 8.0) (see Notes 5 and 6).
Table 1
Seeding density and volume of medium for each well in different plate sizes
Tissue culture
plate
Cell seeding density
a
(cells/well)
Medium volume
(ml/well)
Transduction medium volume
(ml/well)
48-well plate
2.5 Â 10
4
0.3
0.15
24-well plate
5 Â 10
4
0.5
0.25
12-well plate
10 Â 10
4
1.0
0.50
6-well plate
30 Â 10
4
3.0
1.50
a
The values reported in table are example seeding values and may need to be adjusted based on a particular cell line or
desired cell density during transduction
Surface Modification of Adenovirus Vector
361
the volume of virus required to produce the desired MOI.
General formulae:
Virus
IU
well
À Á ¼ seeding density
cells
well
À Á Â MOI
IU
cell
À Á
Vol:of Virus
μl
well
¼
Virus IU=well
ð
Þ
Viral titer IU=μl
ð
Þ
Example: Calculating the amount of virus required in a 24-well
plate.
I. Amount of virus needed for transduction:
Seeding density ¼ 5 Â 10
4 cells
well
À Á
; MOI ¼ 200
Virus
IU
well
À Á ¼ 5 Â 10
4 cells
well
À Á Â 200
IU
cell
À Á ¼ 1 Â 10
7 IU
well
À Á
II. Transforming IU into volume of virus needed per well:
Viral titer ¼ 1 Â 10
10 IU
ml
À Á ¼ 1 Â 10
7 IU
μl
;
Virusð
IU
well Þ ¼ 1 Â 10
7
ð
IU
well Þ
Therefore,
Vol:of Virusð
μl
well Þ ¼
1Â10
7 ð
IU
well Þ
1Â10
7 ð
IU
μl Þ
¼ 1ð
μl
well Þ
As a result, 1 μl of the viral stock is needed to transduce 5 Â 10
4
cells with a MOI of 200.
3.3 PEGylating
Adenovirus
The following procedure is designed to produce 10
7 CPP-PEG-Ad
particles, which in our case is suitable for transducing one well of a
24-well plate. The reaction can be scaled to transduce a greater
number of wells or a larger plate.
1. Dilute 1 μl of virus from the purified viral stock into 99 μl of
50 mM HEPES buffer (pH 8.0) to reach 100 μl. Keep the virus
on ice until used.
2. Prepare 1 ml of stock solution of 1 mg/ml MAL-PEG-NHS
(5 kDa) in 50 mM HEPES buffer (pH 8.0) (see Notes 5 and 6).
Table 1
Seeding density and volume of medium for each well in different plate sizes
Tissue culture
plate
Cell seeding density
a
(cells/well)
Medium volume
(ml/well)
Transduction medium volume
(ml/well)
48-well plate
2.5 Â 10
4
0.3
0.15
24-well plate
5 Â 10
4
0.5
0.25
12-well plate
10 Â 10
4
1.0
0.50
6-well plate
30 Â 10
4
3.0
1.50
a
The values reported in table are example seeding values and may need to be adjusted based on a particular cell line or
desired cell density during transduction
Surface Modification of Adenovirus Vector
361