Please find the following protocol:

Infection of Cells with Retroviruses


Overview

The easiest method is to infect cells in 24-well dishes. This will enable you to titrate the virus in question and will result in individual infected clones.


Virus Infection of Cell Line

1. Prepare the cells for the day of infection by growing in
microtiter plates until the cells are approximately 50%
confluent (or slightly less).

2. Prepare dilutions of virus stock in DME Dilution Solution (e.g.,
undiluted, 1:5, 1:10, 1:100, 1:1000).

3. Aspirate the media from each microtiter plate and add 200 μl
of the virus dilutions prepared in Step #2.

4. Incubate at 37°C in a CO2 incubator for 5 to 6 hours.

5. Add 1 ml of DME Complete Media to each well.

6. Incubate the cells for two full days then discard the media.

7. Trypsinize each well by adding 250 μl of DME with Trypsin,
mix gently, and incubate at room temperature for a couple
of minutes (or until the cells dislodge).

8. Collect the cells with a large bore sterile pipette tip and split
the cells into 100 mm Petri dishes.

9. Place cells into Selection Media.

10. Change the Selection Media every 3 to 4 days and watch for
positively infected clones.



Collection of Infected Cells

1. Aspirate the media from the plate, add an equal volume of
sterile TE buffer, and mix gently.

2. Aspirate the TE Buffer from the plate. Repeat the TE Buffer
wash 2 more times (Steps #1 and #2).

3. Place a sterile Cloning Cylinder down on a single clone.

4. Add 3 drops of pre-warmed (37°C) of sterile DME with
Trypsin.

5. Incubate at room temperature for approximately 5 min.

6. Gently pass the trypsinized cell suspension up and down
between 5 to 10 times with a P200 pipette set to 100 μl.

7. Transfer the cell suspension into each of 2 wells of a 24-well
microtiter plate.

8. Add selection media and grow appropriately.


Solutions

TE Buffer...................................................................Sterilize
....................................................10 mM Tris, pH 8.0
...............................................................1 mM EDTA
DME with Trypsin......................................0.05% (w/v) Trypsin
......................................DME Complete Media
Pen Strep Solution (10X).................1000 Units/ml Streptomycin
............................1000 μg/ml Penicillin
DME Dilution Solution...................................10 g/ml Polybrene
................................DME Complete Media
DME Complete Media..............................1X Pen/Strep Solution
......................10% (v/v) Fetal Calf Serum
......................................Prepare in DMEM


BioReagents and Chemicals

Tris
Streptomycin
Penicillin
EDTA
Polybrene
Fetal Calf Serum (FCS)
DMEM
Trypsin


Reference Link

http://www.bio.com/protocolstools/protocol.jhtml?id=p647