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What constitutes a colony for functional titer determination?

It's difficult to provide guidance without knowing how the 'colonies' look, but we can try to clarify our expectations: We usually choose a cell culture well (or wells) with relatively few GFP+ cells for counting. This makes it much easier to distinguish unique colonies. Remember that we are trying to determine how many unique transduction events occurred on the day of transduction. This means we want to count cells that received only one viral particle. The number of cells we expect to see in a single colony will depend on the cell cycle length of the cell line and the amount of time that has passed between the transduction and the time of counting. For example, HEK293T cells typically undergo 3-4 rounds of cycling in 72 hours. So from one initial transduction event, we might expect approximately 8 GFP+ cells in a single colony. It's common to see colonies with more or fewer cells, but they are typically around that size. If you are having trouble distinguishing unique colonies, we would suggest counting a well corresponding to a more dilute sample of virus.