17. Add 200 μL of each sample to the LumaCyte 96-well plate.
Place the plate in the Radiance instrument for 15 min before
initiating analysis. Please refer to the Radiance +Autosampler
Quick Start Guide or User Manual for additional details on
operating the Radiance instrument and data analysis. Either
the media-only or serum-only controls will be considered 100%
neutralization, while the virus-only controls will be considered
0% neutralization. The neutralizing values of the test samples
containing virus and serum will be determined based on where
they fall between these two bounding values.
18. Store cells at 4
C if not analyzing immediately. Do not freeze.
4 Notes
1. If working with adherent cultures, cells will need to be
detached using an appropriate dissociation buffer. In general,
a gentler enzyme-based preparation such as TrypLE or Accutase yields better results than standard Trypsin.
2. A strong correlation can be obtained with various types of cell
culture media, including serum-containing and serum-free. It
is recommended to be consistent with the medium that is used
for growth or infection whenever possible as this can help
minimize changes as a result of media composition rather
than viral infection.
3. A viral stock of known titer is needed to create a correlation
between Radiance measurements and a traditional viral infectivity assay. In general, a correlation can be made between
various types of assays, including TCID50, plaque assay, or
fluorescent foci assays.
4. Fixation of cells with paraformaldehyde can in some cases
improve the consistency of results. It is best to determine this
empirically for a given cell and virus combination.
5. 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 condition, including MOI 0 as a negative
control, for each time point, as well as three wells to count prior
to infection. When developing an initial LFC-based assay, use
the incubation time of the plaque or TCID assay as a guide. In
general, the time post infection can be shortened 3–5 times.
Thus, a 6–8-day plaque assay can generally be shortened to
2 days with an LFC-based assay.
6. If desired, cells can sometimes be seeded and infected on the
same day. In this case, the cell density should be increased and
the cells allowed to attach for an appropriate period of time,
Viral Infectivity Analysis Using Laser Force Cytology
583
Place the plate in the Radiance instrument for 15 min before
initiating analysis. Please refer to the Radiance +Autosampler
Quick Start Guide or User Manual for additional details on
operating the Radiance instrument and data analysis. Either
the media-only or serum-only controls will be considered 100%
neutralization, while the virus-only controls will be considered
0% neutralization. The neutralizing values of the test samples
containing virus and serum will be determined based on where
they fall between these two bounding values.
18. Store cells at 4
C if not analyzing immediately. Do not freeze.
4 Notes
1. If working with adherent cultures, cells will need to be
detached using an appropriate dissociation buffer. In general,
a gentler enzyme-based preparation such as TrypLE or Accutase yields better results than standard Trypsin.
2. A strong correlation can be obtained with various types of cell
culture media, including serum-containing and serum-free. It
is recommended to be consistent with the medium that is used
for growth or infection whenever possible as this can help
minimize changes as a result of media composition rather
than viral infection.
3. A viral stock of known titer is needed to create a correlation
between Radiance measurements and a traditional viral infectivity assay. In general, a correlation can be made between
various types of assays, including TCID50, plaque assay, or
fluorescent foci assays.
4. Fixation of cells with paraformaldehyde can in some cases
improve the consistency of results. It is best to determine this
empirically for a given cell and virus combination.
5. 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 condition, including MOI 0 as a negative
control, for each time point, as well as three wells to count prior
to infection. When developing an initial LFC-based assay, use
the incubation time of the plaque or TCID assay as a guide. In
general, the time post infection can be shortened 3–5 times.
Thus, a 6–8-day plaque assay can generally be shortened to
2 days with an LFC-based assay.
6. If desired, cells can sometimes be seeded and infected on the
same day. In this case, the cell density should be increased and
the cells allowed to attach for an appropriate period of time,
Viral Infectivity Analysis Using Laser Force Cytology
583