9. If large cell aggregates or clumps are expected, filter cells
through a 50–70 μm filter or cell strainer.
10. Centrifuge the cells for 5 min at a speed of 150–200 Â g (see
Note 10).
11. 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).
12. 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. The Radiance measurements will be correlated with the initial amount of
virus added to each condition (i.e., the MOI).
13. Store cells at 4
C if not analyzing immediately. Do not freeze.
14. Once an initial virus titer correlation has been established
between the Radiance and traditional methods, the Radiance
software can be programmed to provide virus titer measurements in subsequent assays.
3.2 In-Process
Infectivity Assay
with Sf9 Cells
(Real-Time Titer)
1. Seed cells following standard cell culture techniques. For Sf9
suspension cells, seed cells in a 125 mL shake flask at
800,000 cells/mL in 30 mL growth medium at 26–28
C
and 125–150 rpm.
2. Culture cells until they have reached a density between
1,000,000–2,000,000 cells/mL and transfer cells to 6-well
plates at 500,000 cells/mL in 3 mL. After 2 h, infect at a
range of MOI values using a virus stock of known titer. 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 (see Notes 5 and 8).
3. At the desired time points (generally 1–3 days post infection),
determine the cell density of 2–3 different conditions and
harvest a sufficient volume to achieve a concentration of
~600,000 cells/mL (see Note 9).
4. Centrifuge cells at a speed of 200 Â g for 5 min.
5. For each condition, save a separate sample to determine the
titer using the comparative method. If the virus is secreted, save
a supernatant sample. If the virus is cell-associated, save a
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Colin G. Hebert et al.
through a 50–70 μm filter or cell strainer.
10. Centrifuge the cells for 5 min at a speed of 150–200 Â g (see
Note 10).
11. 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).
12. 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. The Radiance measurements will be correlated with the initial amount of
virus added to each condition (i.e., the MOI).
13. Store cells at 4
C if not analyzing immediately. Do not freeze.
14. Once an initial virus titer correlation has been established
between the Radiance and traditional methods, the Radiance
software can be programmed to provide virus titer measurements in subsequent assays.
3.2 In-Process
Infectivity Assay
with Sf9 Cells
(Real-Time Titer)
1. Seed cells following standard cell culture techniques. For Sf9
suspension cells, seed cells in a 125 mL shake flask at
800,000 cells/mL in 30 mL growth medium at 26–28
C
and 125–150 rpm.
2. Culture cells until they have reached a density between
1,000,000–2,000,000 cells/mL and transfer cells to 6-well
plates at 500,000 cells/mL in 3 mL. After 2 h, infect at a
range of MOI values using a virus stock of known titer. 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 (see Notes 5 and 8).
3. At the desired time points (generally 1–3 days post infection),
determine the cell density of 2–3 different conditions and
harvest a sufficient volume to achieve a concentration of
~600,000 cells/mL (see Note 9).
4. Centrifuge cells at a speed of 200 Â g for 5 min.
5. For each condition, save a separate sample to determine the
titer using the comparative method. If the virus is secreted, save
a supernatant sample. If the virus is cell-associated, save a
580
Colin G. Hebert et al.