PLAQUE ASSAYS FOR ADENOVIRUS TITRATION

-Set up 60 mm dishes of P11 cells to be 100 confluent at time of infection.

 

-Remove medium from dishes, add 0.2 to 0.5 ml virus and adsorb for 30 � 60 minutes at room temperature, rocking dishes occasionally.  For titration, test dilutions of 10-5 to 10-11.

 

-Add 10 ml complete DMEM-agarose overlay per plate and let cool.  Add 1 ml normal P11 growth media to top so as not to allow agarose to dry out.  Incubate at 37°

 

-Plaques should be visible within 4 � 5 days and should be counted for titration at day 7 and day 10.

 

To make complete DMEM-agarose:

 

-Prepare 2X DMEM. 

 

-As needed prepare 2X DMEM plus additives:  to 82 ml 2X DMEM add 10 ml Horse serum, 2 ml L-glutamine, 2 ml pen-strep, 2 ml fungizone and 2 ml autoclaved 5 yeast extract and sterile filter

 

-Autoclave 1 g agarose in 100 ml water.  Microwave prior to use and keep at 45 � 50° water bath until use.

 

-Prior to adding to plates make a 1:1 mixture of the agarose and complete DMEM