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Adenovirus infection, MOI and Transduction Efficiency - (Mar/21/2013 )

I am trying to determine the optimal MOI for adenovirus infection of epithelial cells. Adenovirus will be expressing GFP.

1. MOI range - 0, 10, 100, 500, 1000?

2. How soon should I look for GFP expression after infecting cells and how long should I continue to look at GFP?

3. When evaluating GFP with fluorescent scope, scan a few fields for each MOI and find the lowest MOI with full GFP expression for the optimal one?

4. Comments about infecting epithelial cells:
-confluency at time of infection, 80-90%?
-serum/no serum? If no serum, should I include all other compenents - insulin, antibiotics, etc?
-reduce amount of media for infection (i.e. 10cm plate, normally 10ml but use 3-5ml for infection)?
-length of time to infect, overnight then change media?



I think you will find that you may need to abbreviate that range significantly to get any meaningful data. An MOI of over about 50 will kill the cells just from the viral load..

The easiest way to do this may be to do it with a fluorescent plate reader. - look for maximal signal with each moi at defined time points