example, in the case of adherent HEK-293 cells (ATCC
®
CRL-1573) and TrypLE cultured in a T-75 flask:
(a) Aspirate the cell culture medium and then gently wash the
cell monolayer with 5 mL of DPBS.
(b) Remove the DPBS and add 3 mL of TrypLE.
(c) Incubate for 5 min at 37
C or until the cells have visibly
detached.
(d) If needed, gently tap the vessel to dislodge the cells.
(e) Add an appropriate volume of cell culture medium to
resuspend the cells, pipetting up and down to break up
any clumps that are present.
3. Once the cells are resuspended, perform a count in order to
determine the dilution required.
4. Seed cells at 150,000 cells/well in a 24-well plate (see Note 5).
5. Incubate overnight (see Note 6), and then infect cells the
following day using the standard infection procedure for that
virus (see Note 7). Using a virus stock with known titer, infect
cells with a range of MOI values (see Note 8). Start with an
MOI as high as practical (typically between 1 and 10) and infect
with an additional 5–6 MOI dilutions. For example, infect with
MOI values of 1.0, 0.1, 0.01, 0.001, 0.0001, and 0.00001. If
infecting with an initially unknown stock, it is recommended to
start with a dilution of 10
À2 and 6 additional dilutions. Sacrificing one or two wells in order to get an accurate cell count
prior to infection is recommended to improve the accuracy of
the calibration curve.
6. Each day following infection, observe the cells. Once there are
visible cytopathic effects at the highest virus concentration,
harvest the cells at all MOIs as detailed below and analyze
using Radiance.
7. On the day of harvest, detach cells using TrypLE or Accutase
per the manufacturer’s protocol (e.g., with 200 μL of dissociation solution per well of a 24-well plate, incubating for
5–7 min). In some cases, it is beneficial to save the cell supernatant rather than discarding it as it may contain floating
infected cells. The supernatant can be combined with the dissociated cells to create a complete picture of the infected cell
population.
8. If the cell concentration is expected to be >1,000,000 cells/
mL, it is recommended to count the cells after they have been
harvested to determine the volume of LumaCyte Stabilization
Fluid required to dilute the cells to an optimal concentration of
~600,000 cells/mL (see Note 9).
Viral Infectivity Analysis Using Laser Force Cytology
579
®
CRL-1573) and TrypLE cultured in a T-75 flask:
(a) Aspirate the cell culture medium and then gently wash the
cell monolayer with 5 mL of DPBS.
(b) Remove the DPBS and add 3 mL of TrypLE.
(c) Incubate for 5 min at 37
C or until the cells have visibly
detached.
(d) If needed, gently tap the vessel to dislodge the cells.
(e) Add an appropriate volume of cell culture medium to
resuspend the cells, pipetting up and down to break up
any clumps that are present.
3. Once the cells are resuspended, perform a count in order to
determine the dilution required.
4. Seed cells at 150,000 cells/well in a 24-well plate (see Note 5).
5. Incubate overnight (see Note 6), and then infect cells the
following day using the standard infection procedure for that
virus (see Note 7). Using a virus stock with known titer, infect
cells with a range of MOI values (see Note 8). Start with an
MOI as high as practical (typically between 1 and 10) and infect
with an additional 5–6 MOI dilutions. For example, infect with
MOI values of 1.0, 0.1, 0.01, 0.001, 0.0001, and 0.00001. If
infecting with an initially unknown stock, it is recommended to
start with a dilution of 10
À2 and 6 additional dilutions. Sacrificing one or two wells in order to get an accurate cell count
prior to infection is recommended to improve the accuracy of
the calibration curve.
6. Each day following infection, observe the cells. Once there are
visible cytopathic effects at the highest virus concentration,
harvest the cells at all MOIs as detailed below and analyze
using Radiance.
7. On the day of harvest, detach cells using TrypLE or Accutase
per the manufacturer’s protocol (e.g., with 200 μL of dissociation solution per well of a 24-well plate, incubating for
5–7 min). In some cases, it is beneficial to save the cell supernatant rather than discarding it as it may contain floating
infected cells. The supernatant can be combined with the dissociated cells to create a complete picture of the infected cell
population.
8. If the cell concentration is expected to be >1,000,000 cells/
mL, it is recommended to count the cells after they have been
harvested to determine the volume of LumaCyte Stabilization
Fluid required to dilute the cells to an optimal concentration of
~600,000 cells/mL (see Note 9).
Viral Infectivity Analysis Using Laser Force Cytology
579