Infection with Retrovirus
A. Preparation of retrovirus
1. Thaw frozen PA317 cells (packaging cells producing retrovirus) onto 2 x 150 mm
plates in complete medium.
2. Once the cells are confluent, split one of the plates 1:4 onto a 150 mm plate. The
other plate should be frozen down in 90% Serum 10% DMSO for future use.
3. Once the passaged cells are 70-80% confluent, remove the medium and add 12 ml
of complete medium. This medium should be the medium of the cells that you
want to infect with the retrovirus.
4. Incubate the PA317 cells in the medium for 12-16 hrs.
5. After 12-16 hours, remove infecting media and filter through a 0.45 m filter.
Collect filtered infecting media in 3ml aliquots in 15 ml conical vials, which can
then be frozen and stored at –80oC for up to 6 months. NOTE : THE
SUPERNATANT IS NOW CONSIDERED INFECTIOUS AND ALL
LABWARE THAT COMES INTO DIRECT CONTACT WITH IT SHOULD BE
IMMEDIATELY TREATED WITH BLEACH AND THEN DISPOSED OF IN
THE BIOHAZARD WASTE
6. Another collection of viral supernatant can be done simply by repeating steps 3-5
for the PA317 cells still on the plate.
B. Preparation of cells for infection
1. If cells are being thawed, passage the cells once or twice before infection to
ensure viability.
2. Once the cells are 40-60% confluent on a 100 mm plate, add 3 ml of infecting
media with 3 ml of fresh media with a final concentration of 4g/ml polybrene to
the cells. Incubate the 10mm plate with 6 ml of media for 12-16hrs (O/N).
3. Aspirate off the infecting media and replace with fresh medium, incubate for 7-8
hrs (during the day).
4. Once again, add 3 ml of infecting medium with 3 ml of fresh media with a final
concentration of 4g/ml polybrene to the cells and incubate them for 12-16hrs
(O/N).
5. Aspirate off the infecting medium and replace with fresh media, follow
proliferation of cells for 48 hrs.
6. Once cells are confluent split the cells 1:3 onto 2 x 100 mm plates. One of the
plates will be used for selection, the other will be a back up in case a problem
arises in the selection process.
C. Determination of Optimal Dose for Selection
1. With uninfected cells determine the number of cells per cm2 at confluency.
2. In each well of a 6-well dish (8cm2/well), plate out the uninfected cells at 20-40%
confluency and allow them to attach O/N.
3. Set up the following dilutions in media in the 6 wells :
Puromycin (ng/ml)
50
100
250
500
750
1000
4. The optimal dose of puro will be the dose at which all of the uninfected cells were
dead three days after the introduction of the drug.
D. Selection of Infected Cells
1. With two 100 mm plates split at 1:3, place one under selection using the optimal
drug dose discovered above and leave one as a back up.
2. To the plate undergoing selection, add medium containing the correct dilution of
drug to the cells and incubate for 3-4 days for puro.
3. If numerous dead cells appear in the medium during selection, it should be
replaced with fresh medium with the appropriate drug dose.
4. At the end of selection, aspirate off the drug containing medium, wash the cells
with Sol’n A (or medium) and place them back on their regular medium without
drug.
5. To ensure all the uninfected cells on the infected plate are killed off by the drug
during selection, a parallel “kill plate” can be set up. A kill plate is a plate of
completely uninfected cells which undergo selection exactly as a plate of infected
cells would. At the end of selection, all of the cells on the kill plate should be
dead.