Is there a protocol for titering lentivirus by flow cytometry (FACS)?

The formula that most groups use for calculating titer by flow cytometry is below: titer = {F X (Co/V)} XD F is the frequency of GFP-positive cells as determine by flow cytometry Co is the total number of target cells infected V is the volume of the inoculum D is the virus dilution factor Customers should be careful when choosing a dilution to use for flow analysis. Ideally the cells should be somewhere between 2-20% GFP+ to make sure that they are not counting cells that received multiple copies of the virus. Ideally they would analyze a dilution series and pick the dilution that falls into this range for calculating the titer. It is possible to get a different titer from flow cytometry compared with counting under a microscope. This depends on the flow cytometer settings and the sensitivity of the particular cytometer. Customers need to set the gates for each run by using non-transduced cells and cells transduced with a high enough MOI such that nearly 100% of cells are GFP+.