Procedure: Day One 1. Plate cells in 60 mm dishes using 5 ml DMEM containing 10% Calf Serum. The protocol has worked well for NIH3T3 with 1 x 105 cells and BALB/c3T3 with 2 x 105 cells. For the NIH cell line use 10% fetal calf serum and for the BALB cells use 10% calf serum.

2. Grow cells overnight under appropriate conditions.

Day Two

1. Thaw virus stocks at 37°C and as soon as it has thawed transfer to ice bath.

2. Prepare serial dilutions of virus stock in ice-cold DME containing 10% calf serum (prepare dilutions just before use):

a. 1:10 (150 μl virus stock + 1,350 μl media)

b. 1:100 (150 μl of 1:10 virus stock + 1,350 μl media)

c. 1:1,000 (150 μl of 1:100 virus stock + 1,350 μl media)

d. 1:10,000 (150 μl of 1:1000 virus stock + 1,350 μl media)

e. control (1500 μl media).

3. Add 7.5 μl of Polybrene Stock (final concentration 4 μg/ml) into diluted virus stocks.

4. Remove media from cell culture.

5. Immediately add 500 μl virus stock drop-wise while rocking gently onto the cells. Do at least duplicate plates per virus stock serial dilution.

6. Incubate for 2 to 3 hours in a cell culture incubator at 37°C at 95% Humidity and 7% CO2.

7. Add 5 ml of DMEM media containing 10% calf serum and continue incubation.

For Neo Viruses

1. Day 4 (two days of incubation with virus stock) harvest cells and resuspend in DMEM media containing 10% calf serum.

2. For each viral stock dilution, plate 250 μl into 2 separate cell culture dishes. If you prepared duplicate plates per virus stock then there now should be four (4) plates per viral dilution.

3. Add 5 ml DMEM containing 10% calf serum.

Solutions

Polybrene Stock.......................................800 μg/ml Polybrene DMEM Media......................10% Fetal Calf Serum or Calf Serum .........................................................DMEM Media

Bioreagents and Chemicals:

Fetal Calf Serum (FCS) Polybrene DMEM Ice

Contributor: David Bowtell  University of Melbourne, Australia