This is FSC/SSC plot from pancreatic cancer tumor single cell preparation. The gated cells are secreting Interferon gamma so my hunch is its lymphocytes. Aren't lymphocytes supposed to gate at smaller FSC -A or is FSC-a a function of the voltage I chose?
FSC is definitely a function of the voltage you chose. As for the identification of your cell population the use of cell-type specific antibodies against markers is essential. As a starting point I tend to always use an anti-CD45 antibody when analyzing blood or tissue suspensions. It is expressed on all cells of hematopoietic origin except erythrocytes and platelets. Thus it allows a clear differentiation of the immune cells from other cell types present in the tumor.
If cells in the gate release IFN gamma, indeed they are T lymphocytes.
the values for FSC and SSC are not indicators of a given cell population ; in other words, it depends of the cell population analyzed . Depending of the heterogeneity of the cell population, you adjust yourself the profile of cells within the gate.
FSC is a function of voltage chosen and can provide relative size of cells based on light scatter. What antibody panel and gating strategy are you using? How do you perform sample prep, solid state tumors can introduce some unique issues for flow.
You are definitely dealing with Th1 cells in your gated cell mixture ! However, as per your question, activated T-cells might be a minority in the above gated population (and secrete large amounts of IFN-γ). I think that, as mentioned by a colleague earlier, you should label your cells with anti-CD45 antibodies and gate on that population...!
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