Plaque Assays on A549 Cells for Determination of Adenovirus Titers see Notes 13 and

One day prior to plaque assay, A549 cells are plated at 2.0 x 106 cells/ 60-mm dish (Corning, Falcon).

1. On the day of the plaque assay, dishes of confluent A549 cells are washed with 5 mL of serum-free DMEM for 30-60 min prior to addition of the diluted virus; this wash medium is removed immediately before the addition of the virus dilutions.

2. Serial dilutions of virus are made in serum-free DMEM; dilutions are done within a laminar flow hood. Virus is diluted in sterile disposable snap-cap polypropylene tubes, and tubes are vortexed well after each dilution (5-10 s at an 8-9 setting on a 1-10 scale). Typically for CsCl-banded stocks, the initial two dilutions are 1:1000 (10 |L into 10 mL), followed by dilutions of 1:10. Be sure to change micropipet tips after each dilution; avoid contamination of the micropipet barrel (use of barrier tips will help avoid contamination). Care should also be taken to avoid transfer of virus stock on the outside of the micropipet tip by avoiding dipping the tip into the solution, especially in expelling the volume. For CsCl-banded stocks, the range of dilutions that are usually countable is 10-8 to 10-10.

3. A volume of 0.5 mL of the appropriate dilution is placed on confluent A549 cells (each relevant dilution is assayed in triplicate).

4. Dishes are rocked to distribute medium over the monolayer at 10- to 15-min intervals; cells are incubated at 37°C with 6% CO2.

5. During this incubation, microwave the 1.8% Noble agar stock and place this in a 56°C water bath (at least 30 min before overlay). The 2X DMEM (see Note 15) should be warmed to 37°C, and other components of the overlay should be brought to room temperature before mixing.

6. Immediately prior to addition of overlay to the cell monolayers, mix the agar stock with the remaining ingredients.

7. At the end of the 1-h adsorption period, 6 mL of overlay (see Heading 2., item 23) is added to the edge of the dish and the dish is rotated to blend the overlay with the medium used for infection (the 0.5-mL volume of medium used for infection is not removed).

8. Dishes are left at room temperature on a level surface for 5-15 min in order to allow the overlay to solidify.

9. Dishes are then transferred to 37°C and 6% CO2 and incubated for 4-5 d. At that time a second overlay (5-mL/60-mm dish) containing neutral red is added (see Heading 2., item 23). It is important to have a humidified atmosphere in the incubator, but avoid very high humidity because it may cause excess moisture on and around the overlay, resulting in plaques that diffuse excessively and inconsistently.

10. Plaques are first counted 1 d after the neutral red overlay is added (see Note 16). On A549 cells it is not necessary to add more than the first and second overlays.

11. Plaques can be counted at 2- to 3-d intervals until new plaques are no longer becoming apparent. For Ad2 and Ad5 wild-type viruses this may be 12-15 d postinfection; with other serotypes (5,6) and with group C adenoviruses, which have mutations or deletions in the adenovirus death protein (ADP; previously called E3-11.6K), this may be approx 30 d postinfection (7,8). Plaques are most apparent when holding the dish up toward a light source and observing an unstained circular area that has altered light diffraction. Cells initially may not be rounded up and may simply appear unstained, but plaques will typically become more apparent with time.

12. Dishes with 20-100 plaques are used for the calculation of titer (plaque assays are done in triplicate for each of the serial dilutions for more accurate numbers).

13. It is possible to analyze the kinetics of plaque formation to evaluate the virus's ability to lyse the cells at the end of the infectious cycle. To do this, count plaques from the first day that plaques are apparent on any dish/any virus. Do additional counts at 1- to 2-d intervals for all viruses. At the end of the plaque assay (when no additional plaques are becoming apparent [this may be nearly 1 mo postinfection]) calculate the percentage of the final plaque number apparent at each time point during the assay (see Fig. 1).

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