I am still trying to figure out on how to gate lymphocytes population on FLowJo. Attached are 5 plots where all samples are murine spleen and lymph nodes cultured in vitro.
1) My 1st question is based on plots 1,2 and 3 attached here. I am not sure where to place the gates for lymphocytes and live dead population. I was always under the impression that lymphocytes will be from 50k to 200k and live dead within 10^3 on the X-axis. As you can see from the 3 plots, if I change the gate positions I do not see a double population for CD4. Kindly help me in understanding better.
2) My 2nd question is pertaining to plots 4 and 5. Here I am seeing 2 populations on the FSC vs SSC and what I gated on are the lymphocytes I believe. My live dead population here seems a little different. I am not sure if I am actually seeing Live population as it seems really spread out. Here also if I gate my live population within 10^3, I feel my CD4 vs CD69 background is reduced. I am kind of lost with both the analyses. I would really appreciate help with this matter.
Thank you
In flow cytometry, you can never consider that your cells should be somewhere like "50 to 200k" or "10^3" : the cells will be somewhere depending on how you set up your voltages (PMT).
The best thing to do to figure out exactly where your cells are is to create overlap representations. For example you could create a CD3+ cells gate on singlets and then do an overlap of this gate on singlets using a FSC/SSC plot to have a first idea as to where are your lymphocytes. You can do something similar with your viability marker.
@Julien Thank you for the suggestion. Will try that and see how it works.
Dear Achintyan Gangadharan,
I do not think it is very common to use a log scale for side scatter lights (SSC) when you seek only for lymphocytes. Lymphocytes are relatively smooth cells without complex cellular structures and I guess they are more easily gated on the linear scale SSC, although cultured cells could be somewhat different.
For gating FSC and SSC, it might be advised to try microscopic observation of the same culture that would be comparable to the flow cytometry data. It is actually possible to observe forward scatter lights and side scatter lights on microscopy. On microscopy, you may roughly estimate which particle is a lymphocyte and which is not... You might be doing the same on Flowjo but with the digital data instead of the microscopic information. This is because a flow cytometer is actually an automated digital microscope.
In your data, SSC(lo) events are pretty likely to contain most lymphocytes. Then, you might need to clarify whether you need to analyze SSC(hi) cells for the purpose of your study.
Good luck.
For more information,
https://www.bio-rad-antibodies.com/blog/a-guide-to-gating-in-flow-cytometry.html
https://www.researchgate.net/post/Why_are_FCS_vs_SSC_dot_plots_in_linear_scale_and_other_parameters_in_logarithmic_scale
https://www.flowjo.com/learn/flowjo-university/flowjo/getting-started-with-flowjo/59
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