1. Place 5 x 104 cells/well of a 24-well dish the day before titration.
2. On day of titration dilute virus (keep virus on ice as much as possible):
- 100x à 10x (10μl virus in 90μl media)
- 10x à 1x (40μl 10x in 360μl media)*
- 1x à 1/10x (30μl 1x in 270μl media)
- *Transfer 300μl of 1x into a fresh tube
3. Add 2.4μl polybrene to the 300μl of 1x and 1/10x dilutions (final [PB] = 8μg/mL). Mix well.
4. Remove media from well. Add 250μl diluted virus + PB. Remember to add 250μl of plain media to one well for a mock (Don’t add PB to the mock well).
5. Incubate for 2 hours
6. Remove media. Add 1 mL fresh media.
7. After 3 days (Tues à Fri or Fri à Mon) harvest wells
8. Run flow to determine per cent of transfected cells (TF’d cells). Use the data from the dilution (1X or 1/10X) which provided the lowest per cent of Tf’d cells.
Example:
1X: 85 per cent Tf’d cells
Use this à 1/10X: 15 per cent Tf’d cells
9. Calculate the tire based on this equation:
Titre = (1x105 cells) x (per cent of Tf’d cells) x (4) x (dilution factor)
Example:
From flow analysis of 1/10X dilution we found 10 per cent Tf’d cells. What is the titre?
Titre = (1x105 cells) x (per cent of Tf’d cells) x (4) x (1000)
Titre = 4x107 PFU/mL (Don’t worry about the units)