I am studying lymphocytes in the spleen using flow cytometry and I am trying to discriminate doublets in the FSC-A and FSC-H plot. In my data, there are two lines gated as P1 and P2. How should I interpret these data? Can monocytes and lymphocytes form different lines in the FSC-A and FSC-H plots, as I observed P2 to be located to the right of P1 in the FSC-A and SSC-A plots and appeared to be monocytes?
P1 gate is your singlets. P2 gate are those cells that clumped/ doublets.
As for the monocytes and your lymmphocytes, what did you used for your panel? I suggest to add other markers to discriminate several population targets :)
Nam Tae Kim
I appreciate your suggestion to consider the P2 gate as doublets rather than signals.
Actually, I did not use antibodies for monocyte markers because monocytes are not the subject of this analysis.
Based on the position on the FSC-A and SSC-A chart, I assumed that the P2 gate is a monocyte.
I will use only the P1 gate for the following analysis.
I agree with the others. P1 is likely your singlet-gate, and P2 doublets. I only work with human Leucocytes myself, but when looking at T-cells, I now start with SSC/CD3 instead of Lymphocytes (SSC-FSC). I don't want to miss any proliferating cells closer to the Monocytes, the two singlet-gates then take care of the doublets I may end up with. Try to gate your cells of interest before the singlet gate, and then back-gate to se if they end up in your P1-gate.
Lyudmila Akhmaltdinova
Thank you so much for your advice. You're right. It's okay for me to discard monocyte.
Kerstin Andersson
I appreciate your answer.
I wonder how you selected the CD3+ gate in CD3 and SSC-A chart.
It seems that you included the area above the one in which cells are abundant.
> I don't want to miss any proliferating cells closer to the Monocytes
I think I may understand it. You expanded the gate to the SSC that may include the area of the Monocytes?
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