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some help needed for fluorescent focus assay for virus quantitation

virus fluorescent assay

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#1 Andras



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Posted 13 February 2012 - 09:48 AM

I'm trying to set up a virus quantitation system based on fluorescence. I've read about this assay (FFA) first on Wikipedia, but since it wasn't very detailed, I looked up Flint's Principles of Virology (it was given as a reference for the assay).
It wasn't very informative, either. I would like to know how to calculate the FFU using this method.
There are two main options, really: one is to treat the fluorescent signal the same way as in the plaque assay. In this case I'm not really sure how to survey a whole plate for fluorescent signal under a microscope.
The second is to count the number of foci under low mag objective in a few fields, and interpolate. In this case I have the following issues: do you normalize the # of foci to the total # of cells? (After all you can actually count the number of infected cells, and don't need to worry about plaques. In a way it seems like a more accurate way to quantify viruses than the plaque assay.)
Should I count the cells and simply calculate TCID50 based on the number of infected vs total number?

I'm not really sure what to do. Please advise.

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