Dear Tumor Immunology Experts I am trying to isolate Tumor-infiltrating lymphocytes primarily T cells from tumor-bearing mice induced by MC38 cells.
I do not have CD45 beads, but I am using CD45 antibody. I have collected total tumor cells and run the FACS, as you can see the SSC and FSC population in the figure I have attached:
No-1 Is the whole cell population with voltage adjustment to visualize 100 percent cells.
No-2 is also a whole cell population with voltage adjustment to visualize only lymphocytes.
When I go with no-1, the CD8 and CD 4 population percentage are tough to gate even I am using CD45+ cells, but when I am going with no.2, the CD8 and the CD4 population is very easy to distinguish.
It is better if I am using with a no. 2 of voltage, but I am not sure if it can be accepted for publication or not.
I am looking for the valuable advice and guidance.
Thanking you
Dear Prashant,
I have some experience with immunophenotyping MC38 tumors ex vivo.
The way I approach it is to make sure the larger part of all cells (tumor and immune) is in the FSC/SSC plot. Then I gate on alive CD45+ cells, then on CD3+ cells and then on CD8b and CD4. I end up with a pure population of T cells. Remember there are other CD8 and CD4 populations, for example dendritic cells, that are not T cells.
To answer your question; the most important issue is to make sure that you do not exclude your cell population of interest in any gating strategy. As such, I would advise to backgate your T cells to show there are none that fall out of the FSC/SSC gate.
Personally, I do not see a problem in using your second approach (NO-2) as long as you make sure to not exclude any T cells (back gate to make sure).
One small thing, in your second plot, I would not advise to call the gate "lymphocytes" since that gate may contain many other cells.
Hope this helps
Dear Sjoerd Schetters,
Thank you very much for the response. I also have gated CD 45+ cells, but without CD3, As per your suggestion, I will include CD3 this time.
Thanks for suggesting the backgate strategy and leveling out the gate as "lymphocytes."
It was a positive, helpful and informative response.
A big thank you once again
We have one related paper, hope this can help to find out few answers:
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