generally 1–3 h. For same-day infection, the cell count at
infection can be assumed to be the same as the seeding density.
7. Various methods can be used for infection. In some cases, a
medium exchange is required in which case the virus can be
mixed into the replacement media. At other times, the volume
of virus required is sufficiently small that it can be added
directly to the culture. A third variation includes the reduction
of culture medium volume, addition of the viral inoculum for a
set period of time (e.g., 1 h), removal of the virus and addition
of fresh medium.
8. If the precise titer is not known, dilutions can instead be used
and the Multiplicity of Infection (MOI) calculated once the
titer has been determined.
9. Counting the negative control, the highest virus concentration, and a dilution somewhere in the middle will help you
estimate the volumes of stabilization fluid needed for the other
conditions. If the cell count is below the desired concentration
of 600,000 cells/mL, and further concentration through centrifugation is not possible, cells can be resuspended at a lower
concentration keeping in mind the 200 μL volume required to
run on Radiance.
10. The centrifugation speed can be adjusted as needed depending
on the cell line.
11. Stabilization fluid should be at room temperature before adding to cells.
12. It is recommended to prepare a fresh stock every time.
13. In order to develop a method for real-time infectivity determination, a titer must be determined for each sample. This allows
for the correlation between the Radiance data and the titer of
each sample. If desired, correlations can also be made between
other quantities, for example virally driven protein production.
14. The precise cell density can be optimized based on the cell type
and virus. The value in this example represents a cell density
that can be used for next-day infection. Seed sufficient wells for
three replicates per serum condition, including MOI 0 as a
negative control, a serum-only control, and a virus-only control, as well as three wells to count prior to infection.
15. The serum dilutions should be selected based on the particular
assay, but in general an LFC based assay has a larger dynamic
range than a PRNT and thus needs fewer dilutions. For example, where a PRNT might need two- or threefold dilutions, an
LFC based assay can be performed with tenfold serum
dilutions.
16. The neutralization time and temperature can be adjusted as
needed for each particular virus.
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Colin G. Hebert et al.
infection can be assumed to be the same as the seeding density.
7. Various methods can be used for infection. In some cases, a
medium exchange is required in which case the virus can be
mixed into the replacement media. At other times, the volume
of virus required is sufficiently small that it can be added
directly to the culture. A third variation includes the reduction
of culture medium volume, addition of the viral inoculum for a
set period of time (e.g., 1 h), removal of the virus and addition
of fresh medium.
8. If the precise titer is not known, dilutions can instead be used
and the Multiplicity of Infection (MOI) calculated once the
titer has been determined.
9. Counting the negative control, the highest virus concentration, and a dilution somewhere in the middle will help you
estimate the volumes of stabilization fluid needed for the other
conditions. If the cell count is below the desired concentration
of 600,000 cells/mL, and further concentration through centrifugation is not possible, cells can be resuspended at a lower
concentration keeping in mind the 200 μL volume required to
run on Radiance.
10. The centrifugation speed can be adjusted as needed depending
on the cell line.
11. Stabilization fluid should be at room temperature before adding to cells.
12. It is recommended to prepare a fresh stock every time.
13. In order to develop a method for real-time infectivity determination, a titer must be determined for each sample. This allows
for the correlation between the Radiance data and the titer of
each sample. If desired, correlations can also be made between
other quantities, for example virally driven protein production.
14. The precise cell density can be optimized based on the cell type
and virus. The value in this example represents a cell density
that can be used for next-day infection. Seed sufficient wells for
three replicates per serum condition, including MOI 0 as a
negative control, a serum-only control, and a virus-only control, as well as three wells to count prior to infection.
15. The serum dilutions should be selected based on the particular
assay, but in general an LFC based assay has a larger dynamic
range than a PRNT and thus needs fewer dilutions. For example, where a PRNT might need two- or threefold dilutions, an
LFC based assay can be performed with tenfold serum
dilutions.
16. The neutralization time and temperature can be adjusted as
needed for each particular virus.
584
Colin G. Hebert et al.