Infection for Titer ( Click to Download )

All work must be done in the virus hood with appropriate precautions

The day of infection

Plate wells of a six-well plate with 500,000 293T cells per well

Add protamine sulfate to the wells at 5 µg/ml or polybrene at 8 mg/ml (stock is 1000X)

I usually plate and infect at the same time, the cells infect just as well in suspension and the 293T will sit in 30 min or so anyways. This avoids the problem of calculating how many cells you actually infected if you were to plate them ahead (293T grow very fast!).

Add viruses to the wells, add a few dilutions of the virus of interest in duplicate wells.

Well

  1. Mock infected, protamine sulfate but no virus
  2. 10 µl of positive control virus
  3. 10 µl of virus of interest
  4. same as 3
  5. 1 µl of virus of interest
  6. same as 5

Generally these two dilutions will be sufficient to titer lentiviruses made in the lab but if you insert is very small you may get a very high titer virus and you should do an additional dilution of 0.1 µl virus.

Sign up for flow cytometry time 48 to 72 hours post infection (depending on number of samples 15-30 min should be sufficient)

Horizontal transfer test

Take the remaining cells from the wells infected with 10 µl of virus of interest and plate them in a 10 cm dish to carry for horizontal transfer testing

Horizontal transfer testing is done basically to prove these viruses are replication deficient (they are only able to infect cells, not produce other viruses).

1-2 days following plating of leftover infected cells, collect and filter media with a 0.45-micron filter.  Apply media to a fresh plate of 293T cells with polybrene. Allow the media to stay on the cells overnight and then change the media the next day. The following day, either FACS or visually inspect cells for the presence of GFP+ cells indicating replication competent viruses.