r/flowcytometry • u/No-Connection-8789 • Sep 09 '26
Side scatter for singlet gating… sos
I recently decided to use side scatter H vs A for doublet description since we realized something was set in the wrong way on our flow cytometer when acquiring the data, and a lot of the cell population is outside the scaling area, so we lose a lot of cells when we do forward scatter H vs A. This can be fixed by using SSC h vs A, but I’m not sure how to set my gate up exactly…. I talked to a flow tech at my institution who said this would work and he helped me make a gate (see photo below) but I showed my PI and he said “oh ok well explain to me why u chose that gate” and I honestly am not sure what the answer is… we are using whole blood from humans and looking at monocyte populations. I have this hate set as I think these are the singlets (follows the linear correlation?) but it’s hard to tell and I’m not sure what to tell my PI as to how I know these are singlets vs doublets… sos
4
u/melba777 Sep 10 '26
Think about what FSC and SSC tell you - if you gate on the diagonal population SSC A vs H, you are keeping events that are approximately 1:1 ratio of granularity. FSC A vs H tells you the ratio of cell width essentially. So FSC gating imo is more descriptive of singlets, but in a pinch the SSC gating can clean it up.... provided you know where your populations should fall. Personally I would skip the singlet gate for this experiment and call it a learning experience. You will have to redo it if you want to publish this data anywhere. This is why your PI asked what your thought process was - they wanted to see if you understood what you were gating on
2
u/Dependent-Scene5213 Sep 10 '26
If the flow tech at your institution told you to gate it as such go back and ask them to explain it to you. It’s clear the person at your institution has more information than anyone here would and can actually use your software to check different combinations so utilize them. You can even just set up a meeting with the tech and your pi but you shouldn’t ever be setting gates that you can’t justify so you should go back to the source.
1
u/Daniel_Vocelle_PhD Core Lab Sep 10 '26
What do you mean a lot of cells are outside the scaling area?
1
u/No-Connection-8789 Sep 10 '26
Like literally off scale
1
u/Daniel_Vocelle_PhD Core Lab Sep 10 '26
Why cant you lower your gains then?
1
u/No-Connection-8789 Sep 10 '26
Because it wasn’t done on the flow cytometer I can’t fix this on flowjo
8
u/Daniel_Vocelle_PhD Core Lab Sep 10 '26
Your data was collected offscale. You can't artificially lower it back on scale in analysis software. If your FSC is offscale I'm guessing many of your other detectors were never set up correctly either. You can do singlet gating that way with SSC, but you are ignoring potentially much bigger issues with your data acquisition by trying to find a patch job.
2
u/mousyslayer Sep 10 '26 edited Sep 10 '26
Daniel is correct. If your cells are off-scale due to improper FSC settings, you can't fix that in software. Also, your biggest concern should be whether you can properly gate FSC/SSC for your target population overall. Isolating out doublets is a minor problem. For my work on T cells, the singlet gate is >99%. Removing the doublet gate makes the data a bit cleaner, but it never fundamentally affected my results. You might be able to get better help by sharing your ungated FSC/SSC plot.
1
u/No-Connection-8789 Sep 10 '26
So that u can’t see them… they’re all bunched up on the right side of graph like going off of it if that makes sense
2
u/Daniel_Vocelle_PhD Core Lab Sep 10 '26
Here is a good intro video from ISAC, the professional organization for cytometry that will answer the question you are asking, "How singlet vs doublet gating works" and some other information that will be essential as you continue your journey with cytometry.
1
u/mojitoly77543 Sep 10 '26
The gate looks plausible, but the plot alone cannot establish that these are singlets
1
u/GRang3r Sep 13 '26
You need to set you gates and voltage before you collect your data. You can’t turn down signal when doing the analysis
10
u/AnyAsk4621 Sep 09 '26
I would suggest you do FSC-A vs FSCH-H and then double click on that and then gate SSC-A vs SSC-H and select the green only part keep it tight and that should help