Hello all, I am trying to quantify what percentage of infected (GFP+) cells are syncytial (high FSC-H, high Hoechst)via flow cytometry. Cells were infected with a model virus expressing only LASV-GP or mock, and exposed to pH4.5(fusion-inducing) or pH7. My current scheme is to identify the cellular, non-debris population, then from those look at the FSC-H and FITC, then Hoechst vs FITC and conclude that the cells with both high FIT, FSC-H, and Hoechst are syncytial cells. However, while I did tryspinize, it still appears that under SSC-A and SSC-H there are many non-singlets cells, and I am not sure whether these are just clumpy, non-singlet cells, or whether they are syncytial cells. Would perfect syncytia, say 6 just cells fused, show up as non-singlet events?
It's normal to have a certain number of doublets or even triplets in FC. If you think you have above average doublets events on your SSC-A/SSC-H or FSC-A/FSC-H graphs, you can try to adjust your tryspinisation protocol to make it harsher (longer incubation time, higher trypsin concentration).
I don't think you will be able to tell the difference between doublets and syncytial cells on the SSC-A/SSC-H graph.
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