6. Incubate overnight.
7. Prior to infection, count 2–3 wells to determine the amount of
virus that should be added in order to achieve the desired MOI.
8. For each condition of virus and serum samples (not including
the media-only controls, serum-only controls, and virus-only
controls), prepare several serum dilutions in a volume of
100 μL (see Note 15) to be added to a volume of 100 μL
containing the appropriate amount of virus to achieve the
desired MOI.
9. Add each of the 100 μL serum dilutions to a 100 μL virus
sample and incubate for 60 min at 37
C (see Note 16). Prepare
an appropriate 200 μL volume for each control: media only for
the media-only control, serum in media (no virus) for the
serum-only control, and virus in media (no serum) for the
virus-only control.
10. After the neutralization incubation period, remove 200 μL
medium from each well of the 24-well plate(s) prepared in
step 5 and replace it with the 200 μL volume of the control
or test sample prepared in step 9.
11. Incubate the cells at 37
C for the previously determined
amount of time.
12. 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.
13. 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).
14. If large cell aggregates or clumps are expected, filter cells
through a 50–70 μm filter or cell strainer.
15. Centrifuge the cells for 5 min at a speed of 150–200 Â g (see
Note 10).
16. Aspirate the supernatant and resuspend the cells in an appropriate volume of Stabilization Fluid at ~600,000 cells/mL (see
Note 11). If fixation will be necessary, add Paraformaldehyde
to a final concentration of 0.5%. Prepare the mixture of Paraformaldehyde and Stabilization Fluid in a conical tube before
adding to cells (see Note 12).
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Colin G. Hebert et al.
7. Prior to infection, count 2–3 wells to determine the amount of
virus that should be added in order to achieve the desired MOI.
8. For each condition of virus and serum samples (not including
the media-only controls, serum-only controls, and virus-only
controls), prepare several serum dilutions in a volume of
100 μL (see Note 15) to be added to a volume of 100 μL
containing the appropriate amount of virus to achieve the
desired MOI.
9. Add each of the 100 μL serum dilutions to a 100 μL virus
sample and incubate for 60 min at 37
C (see Note 16). Prepare
an appropriate 200 μL volume for each control: media only for
the media-only control, serum in media (no virus) for the
serum-only control, and virus in media (no serum) for the
virus-only control.
10. After the neutralization incubation period, remove 200 μL
medium from each well of the 24-well plate(s) prepared in
step 5 and replace it with the 200 μL volume of the control
or test sample prepared in step 9.
11. Incubate the cells at 37
C for the previously determined
amount of time.
12. 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.
13. 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).
14. If large cell aggregates or clumps are expected, filter cells
through a 50–70 μm filter or cell strainer.
15. Centrifuge the cells for 5 min at a speed of 150–200 Â g (see
Note 10).
16. Aspirate the supernatant and resuspend the cells in an appropriate volume of Stabilization Fluid at ~600,000 cells/mL (see
Note 11). If fixation will be necessary, add Paraformaldehyde
to a final concentration of 0.5%. Prepare the mixture of Paraformaldehyde and Stabilization Fluid in a conical tube before
adding to cells (see Note 12).
582
Colin G. Hebert et al.