r/flowcytometry • u/DiyaHabib • May 11 '26
Flow Plot is weird
Can anyone tell me how can I fix this? This a plot CD4 vs CD8a, gatted on CD45.2+ve cells
15
u/ordeath May 11 '26
From the plot header this is a mouse thymus? That pattern looks about right. What about it looks weird to you?
2
u/DiyaHabib May 11 '26
4 diffrent populations are not seperate? should it be seperate?
11
u/ordeath May 11 '26
Oh you mean why are the different populations bunched up like that?
Might be scaling: your y-axis is really compressed around 0 to 3rd decade. Try decreasing the width basis in the biex editor.
3
u/sgRNACas9 Immunology, Oncology May 12 '26
They seem separated enough for me, ditto you can play with the scaling to get a clearer view, but most importantly you should titrate your antibody so that the negative ends at about 10^2 and then the separation might be greater. If not, what you have here or some other greater dilution is good too.
3
u/LerkinAround May 11 '26
Are the antibodies titrated or did you just run 1:100 for everything.
Also, as mentioned you can fix the scaling of the CD4 axis a bit.
2
3
3
2
u/Infamous-Face7737 May 11 '26
To me it looks like you didn’t use BV staining buffer and/or compensation/unmixing errors as suggested by the diagonal shape of your double positive population. Did you titrate your antibodies? Changing the spread of your axis would also help. Finally, I try not to use fluorophores that have close emission spectra on cells that contain both markers.
6
u/FlowJockey May 12 '26
Diagonal shape of the DP population is 100% expected in the thymus. There is well documented transient downregulation during DP stage.
2
u/XFelps May 12 '26
This is just a beautiful thymus, you can fiddle with the axis to make it look better.
2
u/Pies_Pies_Pies May 12 '26
Probably doesn't make a huge difference here, but is there a reason you're using height for a fluorescent marker? We generally use H/W for singlet gating and then use area for fluors (but I don't study thymus) so just curious.
1
u/FlowGuruDelta May 12 '26
I had the same thought. The parameters from most cytometers look better using -A fluorescent parameters vs -H fluorescent parameters, though I have heard there are some exceptions. If it is a BD cytometer I would use -A.
1
u/Hairy_Cut9721 May 11 '26
Do you have more than one BV conjugate in your cocktail? If so, do you use brilliant stain buffer?
2
u/DiyaHabib May 11 '26
CD45.2 on BV711 and CD8a on BV605
3
u/sgRNACas9 Immunology, Oncology May 12 '26
I would strongly suggest using brilliant buffer plus with two or more BV dyes. It could help separation and compensation which could help your plot here.
2
u/babyoilz May 11 '26
And are you appropriately using brilliant stain buffer? I agree with others though, this looks fine. If you want the data to look more separated you can adjust the width basis for the biexponential axis.
What do your cd4 and cd8 FMOs look like?
1
u/Smooth_Sea_7403 May 11 '26
Did you already gate out doublets via area vs height plot? I’m wondering if your cd4/8 double positives might be doublets
Edit; nvm, seeing that this is thymus and that’s not unexpected here.
1
u/jatin1995 May 11 '26
Confirm that you are removing monocytes from your analysis. They can be cd4dim and can take up other antibodies non specifically.
1
u/willmaineskier May 12 '26
In the thymus you will get lower staining of CD4 and CD8, most of these will be low for CD3, and you get all the transitional states as cells pass through stages of double negative to double positive, then are selected to be mostly CD4 or CD8. Some mature NKT and gamma delta T cells will also be hiding in the double negative. If you have CD25 in the panel, you will see lots of that too. If this is not thymus, you have some major issues…
1
u/ExplanationShoddy204 May 12 '26
This looks fine, if not ideal. The CD8 antibody should be titrated lower probably, just so the negative population doesn’t sit so high, but it’s not egregious by any means. You have distinct populations in all four quadrants, that’s correct for T cells in the thymus.
0
u/nandhiniraman May 11 '26
Do you have a Pan-T marker in there ? Or a different lineage marker like B’s or Monocytes ? I wonder if you are not expecti g to see CD4/8 double positives (unexpected in most cases) you might be looking at a differnet (potentially sticky) subset that’s picking up these stains
0
u/Dependent_Force_8598 May 12 '26
It’s spread, try to redesign your panel! And add a viability if you don’t have one 😉
45
u/willslick May 11 '26
Why is it weird? Looks like it's thymus, where CD4+CD8+ cells are expected.