Hi Raghav,
I had a look at your file, if possible I would first plot FSC-H against FSC-A in order to eliminate the double cells, and then plot the FSC vs the SSC.
You should get a better definition of the populations.
From your FSC vs SSC plot and according to my experience, the monocyte population
is the one above and on the right of the one you selected. But you can easily figure it out by yourself if you plot the FSC vs CD14 staining...
I hope this is useful.
Ciao,
D
It appears you've drawn a region around what is likely the lymphocyte population, and specifically excluded the monocytes. However, I tend not to put much weight into light scatter gating anyway. I would suggest you start with a CD14 vs. CD16 plot (ungated), identify the positive populations (single positives and double positives), and then back-gate on the light scatter plot to assist you in identifying the proper region of the FSC vs. SSC you should gate. CD14 vs. CD16 has a very characteristic profile for normal PBMCs, and if you can identify the populations easily, you should have no problem figuring out what part of the light scatter plot to focus on.
Yes, it appears you have gated on the lymphocyte population. I would suggest adding CD45 to your flow analysis: gate on CD45+ cells, then gate CD14 by CD16 and avoid any forward/side scatter gates. Density gradient centrifugations are not kind to monocytes, so their side and forward scatter properties can increase greatly and monocytes often become artificially activated (increase in HLA-DR surface expression, for example). Due to a variety of detrimental issues involving density gradient centrifugations, we have moved away from isolating PBMCs and now directly stain whole blood. I strongly recommend direct antibody staining of whole blood because we have had much cleaner data and more consistent data. In addition to getting percentages of CD14+CD16+ cells, you can also get cell counts in terms of cells/microliter using fluorospheres if desired.
Best of luck,
Michael
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