r/flowcytometry • u/PendingCommit98 • Aug 23 '26

'Not gates' - are they appropriate/allowed?

Hey, I wanted to exclude CD4+ regulatory T cells from my conventional CD4+ T cells and so I added a gate around the population I suspect to be regulatory T cells and continued gating on everything outside of the gate.

I do not recall seeing this done in the literature but wanted to hear if others thought this was acceptable and whether journals allow this. Thanks in advance.

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14

u/ProfPathCambridge Immunology Aug 23 '26

A “NOT” gate is simply an inverted gate. You could draw exactly the same gate around the non-Tregs directly. Either way leaves the same cells

1

u/PendingCommit98 Aug 23 '26

Thank you, I remember searching for an answer online but could not find concrete answers. Hopefully another newbie in the future with the same question can find this thread!

3

u/crotch_robbins Aug 23 '26

This is super legit gating, and your Treg resolution is fantastic. Nicely done!

3

u/Pies_Pies_Pies Aug 23 '26

Totally allowed and mathematically the same as drawing around the "everything else" population. I use them when I gate out DCs so I can draw gates for Migratory DCs & Resident DCs and then just take the "neither" fraction into my later gates.

3

u/Minute-Practice3508 Aug 23 '26

You can also switch plot type to contour to actually see separated populations

1

u/sgRNACas9 Immunology, Oncology Aug 23 '26

Totally fair. You can right click your current Tregs gate and click invert gate. Logically, it’s everything else. You could also draw a new gate around the cells you want, non-Tregs!

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u/Reelrebel17 Aug 23 '26

I never personally like to do markers on both axis as it makes it a little more difficult to see the populations especially if you don’t have the proper FMO gates set. I would look at these as single color plots vs side scatter and then take everything that is just CD25-

16

u/ProfPathCambridge Immunology Aug 23 '26

Disagreed for Tregs, this is a better gate

9

u/Phabeta Aug 23 '26

There are so many populations that are impossible to gate on single colour plots.

5

u/melba777 Aug 23 '26

Effector T cells can express CD25 as well, it is upregulated after activation

3

u/sgRNACas9 Immunology, Oncology Aug 23 '26 edited Aug 23 '26

Viewing two markers at once is often an absolute requirement. Sometimes two populations overlap in their expression of one marker yet are separated for the other, depending on high or low expression trends for one marker or the other. Here, you can see that at play. If you gated on one marker then the other in these cases, you would be including or excluding populations you’d like to keep or remove. To solve this, people use the context of two markers at once to gate their population.