Biology Reference
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Add 100 μl of streptavidin conjugate per well. Seal with adhesion
foil and incubate for 1 h at 37 °C. Repeat washing step.
Add 100 μl of substrate per well. Incubate for 10 min at room
temperature.
Add 100 μl of stop solution per well to stop reaction.
Measure optical density at 450 nm on a 96-well plate reader. Read
within 30 min.
Calculate the viral titer based on the standard curve from the
AAV-positive controls.
(c) Infectious Center Assay .
The infectious center assay is used to determine the infectious
titer in a rAAV preparation. This assay relies on the ability of
an AAV vector to infect permissive target cells and to replicate
the viral genome; however, it does not reflect the activity of
the transgene. Since rAAV vectors are replication deficient, the
AAV rep gene and adenovirus helper functions are supplied in
trans to ensure amplification of the rAAV genome. Infected
cells are harvested before progeny viruses would be released
and are directly lysed onto a nylon membrane. The DNA is
subsequently hybridized to a specific transgene probe. Each
spot on the film corresponds to an infected cell that has effi-
ciently replicated a single rAAV genome. For the AAV2 vector
serotype, infectivity titers are assessed on an inducible rep/
cap-expressing HeLa cell line (C12 cells) coinfected with dilu-
tions of the rAAV vector together with adenovirus [ 59 ].
The day before infection seed 2 × 10 4 C12 cells per well in a 96-well
plate in 100 μl of DMEM/10 % FBS. Gently shake the plate
to ensure an even distribution of cells across the wells. Incubate
cells overnight at 37 °C.
On the day of infection, dilute 2.5 μl of virus in 247.5 μl of
DMEM-10 % FBS in 96-well plate and perform a series of
tenfold serial dilutions (10 −2 to 10 −9 ) using a new pipette tip
for each dilution.
Thaw wild-type adenovirus type-5 stock on ice. Calculate the
volume of adenovirus required to get a multiplicity of infec-
tion (MOI) of 20 particles forming units (pfu) per cell. Prepare
an adenovirus master mixture to dispense a final volume of
25 μl per well.
Add 100 μl from each serially diluted rAAV and 25 μl of the ade-
novirus master mixture to each well.
Include mock-infected cells and adenovirus-infected cells as negative
controls.
Incubate cells for 40 h at 37 °C.
Resuspend the cells from a 96-well plate by pipetting up and
down several times and transfer the cell suspension into a
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