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[Andras]

Dear All,
I have a few questions about virus titration. As I understand the plaque-assays are in the rage, but all the protocols are different in an ever so slightly way.
I would like to describe how I titrate viruses, and I'd like to ask if it's acceptable.

Cells are seeded on cover slips in 24 well plates.
The virus-containing media is diluted to 10X series and is added for 30 min, then changed to normal media.
After 6 hrs the cells are fixed, and stained for the virus protein.
I take 5 random fields of each coverslip, count the positive and negative cells using CellProfiler, and then calculate TCID50 using an excel spreadsheet I've got.

Someone suggested that this is not good - I should infect the cells with the virus, then collect the media after the first burst of virus production, use this to infect BHK cells, and then do the whole plaque assay-thing, instead of actually counting infected cells. One issue is the whole-plaque forming - Sindbis is supposed to be non-plaque forming virus.

So there are two main issues:
1. is the counting of individual cells permissible instead of plaques? (I can easily count thousands and thousands of cells automatically, in a quite reproducible manner, making statistical analysis a bit more reliable -at least I think.)
2. should I use the two-step approach described above? How do I know when the first bust of viruses are produced? Should I do a time-course for virus production? (RT-PCR/WB of viruses from medium??) I can see why it would make sense to use the same cell type to titrate the virus from all my different cells (HEK, DLD1, etc); yet it would introduce another step were things can go wrong.

Thank you.