Editing Adenovirus Invitrogen (section)

== <br>Adenoviral Stock Titering  ==

1. The day before infection (Day 1), trypsinize and count the cells, plating them such that they will be 80-90% confluent at the time of infection. Incubate cells at 37°C overnight.<br>Example: When using 293A cells, we generally plate 1 x 106 cells per well in a 6-well plate. 

2. On the day of infection (Day 2), thaw your adenoviral stock and prepare 10-fold serial dilutions ranging from 10-4 to 10-9. For each dilution, dilute the adenoviral construct into complete culture medium to a final volume of 1 ml. Do not vortex. 

3. Remove the culture medium from the cells. Mix each dilution gently by inversion and add to one well of cells (total volume = 1 ml). 

4. Swirl the plate gently to disperse the media. Incubate at 37°C overnight. 

5. The following day (Day 3), remove the media containing virus and gently overlay the cells with 2 ml of agarose overlay solution per well. Prepare the agarose overlay solution (enough to overlay one 6-well plate at a time) as described below: 

â¢ For one 6-well plate (2 ml overlay per well), gently mix 12 ml of prewarmed (at 37°C) plaquing media and 1.2 ml of pre-warmed (at 65°C) 4% agarose. Avoid formation of bubbles. 

â¢ Apply the overlay to the cells by gently pipetting the overlay down the side of each aspirated well. Work quickly to prevent premature solidification. 

â¢ Place the 6-well plate in a level tissue-culture hood at room temperature for 15 minutes or until the agarose overlay solidifies. Return the plate to a 37°C humidified CO<sub>2</sub> incubator. 

6. 2 days following the initial overlay (Day 5), gently overlay the cells with an additional 1 ml of agarose overlay solution per well. Prepare the agarose overlay solution as described in Step 5. Allow the agarose overlay to solidify before returning the plate to a 37°C humidified CO<sub>2</sub> incubator. 

7. Monitor the plates until plaques are visible (generally 8-12 days post-infection = Day 10-14). For each well, gently layer the 5 mg/ml MTT solution (1/10 the volume of the agarose overlay) on top of the solidified agar to stain. Make sure the MTT solution is evenly distributed over the entire surface of the well. 

Example: If each well contains 3 ml of agarose overlay, use 300 ÎŒl of 5 mg/ml MTT. 

8. Incubate plates for 3 hours at 37°C. 

9. Count the plaques and determine the titer of your adenoviral stock. 

Vital dye, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue (MTT) as a staining reagent to help visualize plaques. Other vital stains including Neutral Red (Sigma, Catalog no. N7005) are suitable. If you wish to use Neutral Red, prepare a 1% solution (100X stock solution) in water and store at +4°C.<br><br> 

[[Category:Protocols]]