Adenovirus Plaque Assay

1. On the day before titering, split each 100 mm plate of 293 cells to three 60 mm dishes. The number of dishes prepared should be six per virus stock to be titered (two for each dilution to be assayed). Care should be taken to prevent clumps of cells in the monolayer by vigorously dispensing the cell suspension. The dishes should be just confluent when used.

2. On the day of the titering, thaw the virus stock to be titered, Prepare serial 10- or 100-fold dilutions of the virus stocks to be titred in DMEM, 2% FBS and Penicilliiun/streptomycin in 6 ml polystyrene culture tubes in a final volume of 2.0 ml.

3. Aspirate the growth medium from the monolayers of 293 cells.

4. Overlay two 60 mm plates of 293 cells with 0.5 ml of each prepared virus dilution. Record on each dish details of the virus and the dilution of stock.

5. Incubate the cells at 37 oC for 1 hour, rocking at 15 min intervals.

6. Melt 1.3% Noble agar in a microwave and place in a 45 ^oC water-bath for it to equilibrate. Place 2X MEM supplemented with 4% FBS in a 37 oC water bath. Immediately before using, add an equal volume of the molten 1.3% Noble agar to the 2 X MEM.

7. Aspirate infectious virus medium from the cells.

8. Overlay the cells with 6.0 ml of the Noble agar/MEM, 2% FBS. This must be done very gently so as not to disturb the cell monolayer; 293 cells are not strongly adherent, so care is important. Add the agar preparation slowly to the side of the dish. Rock or swirl the dish very gently tp cover the cells and mix in any remaining trace of liquid DMEM. Allow the overlaid cultures to stand at room temperature until the agar solidifies.

9. Incubate the infected plates at 37^oC. Plaques should begin to be visible within about seven days.

10. Count the plaques. Do this as follows:

a) Observe the plates against a dark background, or against fluorescent lighting, whichever makes the plaques most readily visible plaques will appear as white pin-points on the relatively clear background. As the plaques grow, they will appear as larger white spots on the cell lawn and later will form white rings. Cell clumps look macroscopically like plaques at first, but will not grow. Microscopically, plaques are comprised of rounded up cells areas on the monolayer, whereas cell clumps look just as their name suggests as a very dense, raised area of cells.

b) Mark the location of visible plaques with a felt-tipped pen.

c) Count the plaques over a period of several days until the number remains constant.

11. to determine the titer, multiply the number of plaques counted by the dilution factor. Average the results of the serial dilutions.

for example, 1 ul of stock, diluted to 1 ml equals 1:1000 dilution. Infecting with 0.5 ml of the 1:1000 dilution results in a final dilution factor of 1:2000. For large scale purified virus preparations, assaying dilutions of 10^-8, 10^-9 and 10^-10 generally provides an appropriate range.

Protocol 2

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
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)
10−4 dilution: 10 μl of 10−2 dilution + 990 μl 2% medium (=1000 μl)
10−6 dilution: 10 μl of 10−4 dilution + 990 μl 2% medium (= 1000 μl)
10−8 dilution: 10 μl of 10−6 dilution + 990 μl 2% medium (=1000 μl)
10−9 dilution: 300 μl of 10−8 dilution + 2700 μl 2% medium (=3000 μl)
10−10 dilution: 300 μl of 10−9 dilution + 2700 μl 2% medium (=3000 μl)
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