2. Mix 2.4% Avicel solution in water with equal volume of
2Â MEM with 3% FCS, Pen/Strep (1/100), and GlutaMAX
(1/100).
3.4.7 Performing
the Plaque
Reduction Assay
1. Warm maintenance medium and PBS to 37
C in water bath.
2. Remove culture media from a T75 flask containing cultured
Vero cells (see Note 9).
3. Wash cells with 5 mL of PBS and rinse with 2.5 mL of TrypsinEDTA.
4. Incubate flask at 37
C for 5–10 min until monolayer detaches.
5. Add 10 mL of maintenance medium to the flask.
6. Pipet cell suspension up and down at least five times against the
bottom of the flask with a 10 mL pipette. This is to break up the
cell clusters. Be careful not to create air bubbles.
7. Count cells using hemocytometer.
8. Dilute the cell suspension to 3 Â 10
5 cells/mL using prewarmed maintenance medium.
9. If a large number of cells are required for the assay, pool the
cells from several flasks of the same passage number as required.
10. Add 3 mL/well of cell suspension of a 6-well plate and incubate at 37
C in a 5% CO 2 incubator until cells make monolayer
(overnight).
3.4.8 Neutralization
1. Heat-inactivate samples in water bath (56
C for 30 min).
2. Prepare serial fourfold dilution in maintenance medium.
3. Dilute enough volume of virus to 200–240 PFU/mL in maintenance medium.
4. Mix diluted serum sample and virus and incubate at 37
C for
90 min in a 5% CO 2 incubator.
3.4.9 Inoculation of Virus
1. Check plates under the microscope if cells at least 80% of
bottom of the wells.
2. Aspirate cell medium from the wells.
3. Add 0.1 mL per well of neutralized samples per well in
duplicate.
4. Incubate the plate at 37
C for 90 min in a 5% CO 2 incubator
(tilt the plate every 30 min to allow even distribution of the
inoculum in the well).
5. Add 4 mL/well of overlaying media and incubate the plate at
37
C for 6 days in a 5% CO 2 incubator.
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Lei Li et al.
2Â MEM with 3% FCS, Pen/Strep (1/100), and GlutaMAX
(1/100).
3.4.7 Performing
the Plaque
Reduction Assay
1. Warm maintenance medium and PBS to 37
C in water bath.
2. Remove culture media from a T75 flask containing cultured
Vero cells (see Note 9).
3. Wash cells with 5 mL of PBS and rinse with 2.5 mL of TrypsinEDTA.
4. Incubate flask at 37
C for 5–10 min until monolayer detaches.
5. Add 10 mL of maintenance medium to the flask.
6. Pipet cell suspension up and down at least five times against the
bottom of the flask with a 10 mL pipette. This is to break up the
cell clusters. Be careful not to create air bubbles.
7. Count cells using hemocytometer.
8. Dilute the cell suspension to 3 Â 10
5 cells/mL using prewarmed maintenance medium.
9. If a large number of cells are required for the assay, pool the
cells from several flasks of the same passage number as required.
10. Add 3 mL/well of cell suspension of a 6-well plate and incubate at 37
C in a 5% CO 2 incubator until cells make monolayer
(overnight).
3.4.8 Neutralization
1. Heat-inactivate samples in water bath (56
C for 30 min).
2. Prepare serial fourfold dilution in maintenance medium.
3. Dilute enough volume of virus to 200–240 PFU/mL in maintenance medium.
4. Mix diluted serum sample and virus and incubate at 37
C for
90 min in a 5% CO 2 incubator.
3.4.9 Inoculation of Virus
1. Check plates under the microscope if cells at least 80% of
bottom of the wells.
2. Aspirate cell medium from the wells.
3. Add 0.1 mL per well of neutralized samples per well in
duplicate.
4. Incubate the plate at 37
C for 90 min in a 5% CO 2 incubator
(tilt the plate every 30 min to allow even distribution of the
inoculum in the well).
5. Add 4 mL/well of overlaying media and incubate the plate at
37
C for 6 days in a 5% CO 2 incubator.
418
Lei Li et al.