Biology Reference
In-Depth Information
1. Seed HEK-293 cells in a 96-well plate at 60-70 % of conflu-
ency in a total volume of 100 μL/well of infection medium.
2. Twenty-four hours later, prepare serial dilutions of the ten viral
fractions from
step 16
in Subheading
3.2
. Prepare each dilu-
tion in a final volume of 500 μL. Use DMEM supplemented
with 2 % FBS for the dilutions. Typically, start with a 1/10
6
dilution and make 1/10 serial dilutions. Perform 6 dilutions
(
n
= 2 per dilution).
3. Carefully, remove the media from the HEK-293 cells and
infect with 100 μL of each dilution (
see
Note 4
).
4. Two days after infection, count the number of infected cells
using your reporter gene (i.e., GFP or β-galactosidase) or by
immunocytochemistry using the anti-hexon-antibody-based
system as described in Chapter 12. For calculations, consider
only dilutions for wells with less than 10 % of positive cells.
5. Calculate the titer expressed in Infection Units/mL (IU/mL)
using the following equation:
3.3.1 First Titer of the
Viral Fractions
Positive cells Dilution factor
×
×
infectionvolume
(
)
=
TiterIUmL
/
(1)
10
3
6. Pool the three or four most concentrated fractions (usually
fractions 4-7) and aliquot the viral pre-stock (
see
Note 5
) in
0.5 mL tubes and store at −80 °C as quickly as possible.
1. Repeat
steps 1
-
5
from procedure in Subheading
3.3.1
, but
start with a 1/10
9
viral dilution and make ½ serial dilutions as
previously explain. Make 12 dilutions.
2. Calculate the titer applying Eq.
1
. When working with a fluo-
rescent reporter gene and using cells in which the vector is
replicative, select for calculations the most diluted condition
showing positive cells 5-7 days after the infection and consider
the number of positive cells as “1” (
see
Note 6
).
3. Prepare a 1/20, 1/10, and 1/5 viral dilutions in a total vol-
ume of 100 μL. Use 1× PBS Ca
2+
/Mg
2+
plus 0.1 % SDS for the
dilutions (
see
Note 7
).
4. Incubate at 56 °C for 10 min to disrupt viral capsids and release
viral genomes.
5. Centrifuge the tubes at 4,500 ×
g
for 5 min.
6. Measure the optical density of the supernatants at 260 nm
(OD
260
) (
see
Note 8
).
7. Calculate the titer expressed in viral genomes/mL (vg/mL)
using the following equation:
3.3.2 Final Titer
of the Viral Pre-stock
(
)
=
12
11 10
TitervgmLOD ilution factor
/
×
××
.
(2)
260
