1. Seed 1 x 106 cells each well
in two 6-well plates. Allow cells to grow overnight.
No swirling, otherwise cell density will be lower in the center of the well.
For use in the same day, seed 1.5 x 106 each well.
Cells should be 60-80% confluent the day of infection.
2. Label 6 sterile microcentrifuge tubes, to each add 270
ml medium. Add 30 ml
of virus stock to the first tube, mix, and transfer 30 ml
to the second tube. Repeat to make a serial 1:10 dilution of
the virus stock.
3. Remove medium in the 6-well plates using a sterile Pasteur pipette.
4. Add 100 ml of the 10-1
to 10-6 dilution to one of the wells. Repeat
for the second plate.
5. Incubate at room temperature for 1 hour to allow the virus to infect the cells.
6. Melt 10 ml of sterile 2% agarose in H2O (autoclaved)
and keep the agarose molten in 37 °C water bath. Prewarm culture media to
7. Carefully remove the virus without dislodging cells.
8. Mix 1:1 warm culture media and 2% agarose.
9. Gently add 1.5 ml of the 1% agarose to overlay cells in each well. Leave cells
at room temperature for at least 20 minutes. Don't disturb the plates until
the agarose overlay has set.
10. Add 1.5 ml of the culture medium to each well.
11. Incubate the cells at room temperature or 27 °C for 4-5 days.
12. (optional) For virus expressing the beta-galactosidase protein, stain with
X-gal. Remove culture medium, add 1 ml fresh medium + 1 mg/ml X-gal to
each well. Incubate at room temperature or 27 °C in dark for several hours until
blue staining developed.
13. Remove culture medium, add 1 ml of 0.03% neutral red in PBS to each
well. Incubate at room temperature or 27 °C for 2-3 hours.
14. Remove neutral red/PBS, invert the dishes, and leave them in dark
at room temperature overnight. Cells infected with virus are dead, thus
will not take up neutral red as uninfected cells do. Plaques will appear
as small clear area against a red or pink background.
15. Count the plaques on each well and determine the virus titer:
virus titer (pfu/ml) = number of plaques * (1 ml / 0.1 ml) / fold of dilution