1. Seed 0.5-1 x 10⁶ cells in 35 mm tissue culture dishes or 6-well plates. Incubate at room temperature or 27 °C for 1 hour to overnight.

2. Remove medium. Add 100 ml plaque-pick.

3. Incubate at room temperature for 1 hour.

4. Add 2 ml of fresh medium to each dish/well.

5. Incubate at room temperature or 27 °C for 3-5 days.

6. Transfer medium to a sterile tube. Remove cells and debris by centrifugation at 1000 x g for 5 minutes.

7. Transfer supernatant to a fresh sterile tube. This will be the Passage One stock. Store in dark at 4 °C.

Passage Two

8. Seed cells in 100 mm dish (2 x 10⁶ cells / 10 ml) or a 50 ml suspension culture ( 2 x 10⁵ cells/ml ). Incubate at room temperature or 27 °C for 1-2 days.

9. Add 50-200 ml Passage One stock per 10 ml of cells.

10. Incubate at room temperature or 27 °C for 4-6 days.

11. Transfer medium to a sterile tube. Remove cells and debris by centrifugation at 1000 x g for 5 minutes.

12. Transfer supernatant to a fresh sterile tube. This will be the Passage Two stock. Store in dark at 4 °C. For long term storage, aliquot and freeze at -70 °C. Freezing however lowers virus titer.