HI!
I culture RAW cells in DMEM and did flow analysis. When performed flow analysis I saw that there a two populations in the SSC/FSC plot. I believe that the other population was debri. I have attached the image of the log scale and the regular scale, please kindly let me know if the way I have gated to select the cells is right.
Thank you!
Hi Abishai,
It's very possible that your second population contains dead cells. Based on my experience, dead cells generally appear to be smaller when gating SSC and FSC. If you include a viability stain, you can confirm.
Dear Abhishai,
Have you tried doing doublet discrimination? Immunes cells have a tendency to form clumps which may be shown in the FSC plots. Also a live dead exclusion assay using stains like PI/FDA will assure that it is not a dead population. Also, macrophages in different maturation stages can show different morphology and appear as different population in FSC plots so confirm with the maturation markers expression (if they are not dead).
BW
Gargi
Raw cells are macrophage cell line. Like any cells and cell lines one needs to carry out with better cell culture protocol to maintain healthy cells in the culture. Otherwise one will see this double population, one for dying or cellular debris and other with a live population
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