Hello all,
I have performed a number of experiments in differentiating my mESCs, using negative controls (unstained) plus single-stain for each of the Abs used for proper compensation.
Yet, when I finally reach on my last cell gated populations that I would like to sort for further analysis, my backgating shows as the picture depicted. I cannot actually understand if this is normal or not.
My concern lies with the red-marked populations outside the gates. Can you please explain what is the cause of this?
Thank you in advance
The print is too small to read the markers.
Also...how did you mark these gates (just by the negative controls)? I don't even see a clear distinction between your positive and negative markers. When you did the compensation, did you have a clear distinction between your positive and negative? This makes me think that the settings on your flow aren't correct.
Hi Shen-An, thanks for the reply. Sorry, didn't manage to get them larger. The gates have been marked as judged by the negative and positive controls, where there was a somewhat good distinction between negative and positive populations. My question lies on why the backgating shows populations outside my gated areas?
First, I want to make sure that you are using a true parent-child relationship with your gates? And the "red" color is only for the population of the last gate? Usually, for me, since each gate I set has a different color, it's easier for me to trace back linage of my gates for any specific population. So for example, my CD3+CD4+ cells will never show up in the "neutrophil" population of CD45 positive cells.
So... what program are you using? Because the only time I've seen this was a while ago when I was using a free program on the web. Ideally, you would think that when you do subsequent gating, it only gates on the population that you are interested in and it only takes into account of what is visible on your plot. However, I've noticed that it may not be true. If the program doesn't automatically only counts what is visible on the plot inside the gate, and it is counting ALL events inside the gate, even the invisble ones, this is when you will see what you see. Does that make sense?
It's FlowJo, so it's not freeware. I am not sure if the red colour is from the last gate. I assume it is. To be honest, I am getting this backgating "paradox" again mostly on the two last gates (mostly the 5th one) with another project that I am running (human PBMCs and BM-derived cells), so I am not sure if this is a software glitch. The channels affected are not the same between the two projects.
The literature has shown nothing of the kind and I have reset and did the gates from scratch many times.
Can you set your gates to display different colors? You should be able to. Usually the "gate" itself is the color of the population, although it looks like all the gates are black. So I wonder where this red cell population is coming from... I won't think there would be a software glitch that only affected these two experiments.
This is being done on all experiments, not just these ones. I actually moved the last gates to a non-cell position (i.e. on the upper right corner), and the backgating remained the same, which is odd, isn't it?
I will use different colours to see what I will get.
Hi Shen-An, an update on the issue, it has been resolved, flowjo probably has a bug on that issue, since the ancestry is fine. Thanks for your help!
Hello Ioannis,
Thanks for raising this important question, and for your interest in FlowJo for cytometric data analysis.
What you see in that illustration is expected - No, this is not a bug in FlowJo. Backgating is an advanced technique in flow cytometry data analysis, which illustrates a terminal gate of interest overlaid on previous points in the gating hierarchy without regard to that gate being illustrated. In other words the population you see in red is not your final population being back-gated, rather it is a derivative of the final population which does not consider the gate on which it is overlaid.
The fact that a backgate does not consider certain choices along the gating tree, means that it is specifically meant to show you where that gate may be missing potential cells of interest. This can help to refine and better understand gating trees leading to rare subsets that may be difficult or nuanced to define.
TLDR: Backgating is not the same as ancestry.
Hope that helps. If you have further questions on this or other areas of FlowJo, please consider reaching out to the team of flow science and informatics experts, here:
Ian Taylor
BD Life Sciences - Informatics
Dear Ian,
Many thanks for your concise reply. With what you said in mind concerning the cells of interest, how is this defined, as "cells of interest"?
All the best,
Yannis
"Cells of interest" is meant as a generic term to describe any population of events from an FCS dataset which might be used as the starting point for backgating. In other words, a specific population that the researcher is interested in investigating.
Whatever population your gating tree is leading you too, when you run backgating on that subset the resulting overlay of backgates can help to inform and refine your gating strategy.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence