Editing Adenovirus Titering by Anti-Ad Hexon Staining (section)

== Protocol 1 ==

1. Prepare serial dilutions (typically 1/10) of the stock in 96-well dishes using DMEM + 5% FBS. Final amount for each dilution in each well should be 100 mL. Range of dilutions will be selected according to the estimate concentration of the viral stock to titer.

2. Add 50 mL/well of a cell suspension at 1.106 cells/mL (50,000 cells/well) in DMEM + 5% FBS. The cell line must be chosen depending on characteristics of the viral stock to titer (HEK-293 cells are appropriate for most assays) 

3. Incubate virus and cells for 24â48 h (time must be chosen depending on rate of replication of Ad in the cell line in order to avoid secondary infections). For HEK-293 cells incubate<br>for 24â36 h.

4. Remove medium from the wells very carefully to avoid cell loss. Air-dry for 3â5 min.

5. Add 100 uL of 100% ice-cold methanol to each well (fixation). 

Incubate for 10 min at â20°C.

6. Aspirate methanol. Wash each well containing the cells twice with 100 uL of PBS Ca++/Mg++ 1% BSA (PBS without Ca++/Mg++ can also be used, but adding bivalent ions can prevent cells detaching from wells).

7. Add 50 uL of primary antibody diluted in PBS Ca++/Mg++ 1% BSA to each well. For most hybridomas 1/5 dilution from supernatant is recommended. If using a purified anti-adenovirus or anti-hexon Ab, 1/500 dilution can be initially tested.

Avoid bubble formation. Incubate for 1â2 h at 37°C.

8. Wash each well (3Ã) with 100 uL of PBS Ca++/Mg++ 1% BSA.

9. Add 50 uL of FITC or Alexa488-conjugated secondary antibody diluted in PBS Ca++/Mg++ 1% BSA (1/300 dilution is suitable for most commercial antibodies) to each well. Using Alexa488 can increase the test sensibility. Avoid bubble formation. Incubate for 1â2 h at 37°C in the dark.

10. Wash each well (3Ã) with 100 uL of PBS Ca++/Mg++ 1% BSA.

11. Quantify green cells in each well using an inverted fluorescence microscope. A cloud of positive cells (âcomet effectâ) suggests secondary infections and consequently it should be quantified as a single positive cell. For each stock, the mean of different dilutions will be used to calculate concentration.

Note that only 100 uL of each dilution has been used for analysis, so a tenfold factor should be applied to obtain transducing units per milliliter (TU/mL).<br>