Plaque Assay

Plaque Assay Description

1. One day before the assay, plate 293 cells in 6-well plates at a density of ~70%.
2. Serial dilute your virus as follows:

a. Make a 1:100 dilution by adding 10 μl viral stock to 990 μl medium.
b. Starting with a 1:100 dilution, prepare serial 1:10 dilutions by transferring 100 μl of diluted virus to 900 μl medium up to 10⁵- 10¹⁰depend on expected concentration of your viral stock.

3. Infect 293 cells with 0.2 ml diluents of adenovirus. Tip the plates to spread the virus evenly over the monolayer.
4. Incubate the cells for 60 min at 37^oC to allow the virus to infect the cells.
5. During incubation, prepare agarose overlay medium as follows.

a. Melt 5 ml of 5% agarose. Then cool to 44^oC.
b. Warm 50 ml growth medium to 44^oC.
c. Add 45 ml growth medium to 5 ml 5% agarose. Mix well.

6. Remove the virus-containing medium from the cells.
7. Gently add 3 ml of agarose solution to each well, taking care not to dislodge any cells.
8. When the agarose has set, return plates to incubator. Plaques should be visible within 7-10 days.
9. Prepare a 0.03% solution of neutral red in PBS (1 ml 0.33% [w/v] neutral red stock + 10 ml PBS). Add 1ml of the 0.03% neutral red solution to each of the wells and incubate at 37^oC for 2-3 hours.
10. Remove the stain by aspiration, then invert the dishes to allow the plaques to clear.

Note : Neutral red is taken up by healthy cells but not by dead cells. Therefore, plaques appear as clear circles against a red or pink background.

11. Calculate Viral Titer

To calculate the viral titer, count the number of well-isolated plaques. Then use the following formula to determine the titer (pfu/ml) of your viral stock.

#plaques/(dxV) = pfu/ml

d = dilution factor
V = volume of diluted virus/well

Sample calculation:

• An average of 50 plaques formed in the 1:10,000 dilution wells
• Volume of diluted virus added: 0.2 ml

50/(0.0001 x 0.2) = 2.5 x 10⁶ pfu/ml