Editing Adenovirus Plaque Assay (section)

== Protocol 2<br> ==

Adenovirus Plaque Formation Unit Assay

This method measures the plaque formation activity of a virus sample (that is, how many plaques each milliliter of the virus sample can form). It involves<br>infection of permissive 293 cells with the viruses, completion of an infection cycle, and re-infection of neighboring cells to generate a clear plaque. It is the most challenging and the most variable titration method described herein because many factors such as quality of the cells and plaque observations at different time or by different person may affect the results.

1. Plate 293 cells in a 6-well plate at a density of 5 × 10^5 cells/well in 3 ml complete culture media the day before infection.

2. Dilute the virus. A 5-μl aliquot of the virus should be more than enough for this experiment. Make dilutions in 2% medium as following. Mix well after each dilution.

10−2 dilution: 4 μl virus + 396 μl 2% medium (=400 μl)<br>10−4 dilution: 10 μl of 10−2 dilution + 990 μl 2% medium (=1000 μl)<br>10−6 dilution: 10 μl of 10−4 dilution + 990 μl 2% medium (= 1000 μl)<br>10−8 dilution: 10 μl of 10−6 dilution + 990 μl 2% medium (=1000 μl)<br>10−9 dilution: 300 μl of 10−8 dilution + 2700 μl 2% medium (=3000 μl)<br>10−10 dilution: 300 μl of 10−9 dilution + 2700 μl 2% medium (=3000 μl)<br>10−11 dilution: 300 μl of 10−10 dilution + 2700 μl 2% medium (=3000 μl)

3. Infect the cells in the 6-well plate with 1 ml of 10−9 , 10−10 , and 10−11 diluted viruses. Use two wells for each dilution.

4. Add 2 ml of complete culture media 2 h later. Incubate the cells at 37 °C, 5% CO2 overnight (12–16 h).

5. Coat the wells with SeaPlaque agarose + 2× DMEM mixture, 3 ml for each well. Do not add liquid media to the top of the agarose coat.

6. Refresh the cells with 1 ml of SeaPlaque + 2× DMEM mixture every 4 days. Check plaque formation from time to time.

7. Count plaques. Wait for at least 14 days to allow all potential plaques become readily visible under microscope. The wells that contain well-isolated plaques should be counted.

8. Calculate the titer (pfu/ml). For each well, the titer (pfu/ml) = the plaque count × the dilution factor. Take average of the titers obtained from each well as the PFU titer of the virus sample<br>

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