Is there a difference in titer when titering virus by FACS versus microscopy?
Yes, determining titer by FACS will always give you a lower titer when compared with direct counting of GFP positive cells/colonies by fluorescence microscopy. On average there is approximately a log difference between the two methods. The reason for this discrepancy is that it is very difficult for FACS to distinguish weakly GFP positive cells from background. The weakly expressing cells will get gated out. Other factors that may influence the titers include the cell type used for titering, the use of polybrene during transduction, the volume of culture media and concentration of serum during transduction, and the length of the transduction period.