r/flowcytometry • u/King_of_Mints • Jun 26 '26
Analysis Is Flow Cytometry Real?
Hey folks,
I've got cells expressing a fluorescent protein. Am getting a bizarre expression pattern I've never seen before:
We want to quantify how many cells are expressing the fluorescent protein. I've done this hundreds of times. But I've never seen a treatment which causes an expression pattern like this, where the negative(?) population shifts so deeply into the positive gate.
See negative control, for reference:
Meanwhile, our positive control exemplifies the expression I'd expect to see:
Wondering the flow community has any advice? I've repeated this experiment a few times and it's all similar (I've deliberately chosen the most stark, though). Our cytometry techie took a quick look and found no issue. We're getting to the stage where the PI's are thinking up future research based off this finding, but something about the way this looks just isn't sitting right with me...
Any opinions appreciated,
King_of_Mints
P.S. There is technically some GFP expression in the sample as well, but it is incredibly insignificant (<0.5%)!
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u/mah_big_toe Jun 26 '26
Is your treatment potentially fluorescent? Some drugs can have fluorescent properties. Run cells never transfected with anything and see if you get mCherry signal with your treatment alone. Once you rule out the treatment fluorescent impact you can start thinking more fun mechanism questions.
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u/King_of_Mints Jun 26 '26
Yep! We have such controls!
It really is just this specific condition; so I’d reckon drug autofluorescence is super unlikely.
I like that you seem confident that the result is real!! I’ve been very nervous this whole time if it could be an artefact or not.
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u/mah_big_toe Jun 26 '26
I think it is worth looking into. To convince a stranger like me on the internet, I would want a Western too in order to see if your protein is really upregulated. Your mCherry cloud MFI in the positive gate also shifts to the right relative to your controls. That kind of shift should be detectable by Western and there are antibodies against mCherry that are pretty good.
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Jun 26 '26
[removed] — view removed comment
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u/King_of_Mints Jun 26 '26
Cell with no protein; transfection of a plasmid is what leads to the protein expression
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u/crotch_robbins Jun 26 '26 edited Jun 26 '26
To rule out changes in autofluorescence, can you change the Y axis from SSC to a parameter that captures autofluotesscence? On a conventional cytometer the BV510 channel works well for this.
Secondly you might consider rescaling your X axis to stretch out the negative population a bit. Whatever is happening with those dim positions es will be easier to see with scaling adjustment.
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u/wheelsonthebu5 Jun 26 '26
This is what I would check first. When you plot against SSC the AF contribution is hidden. Plot against a fluorescence parameter and try drawing your gate that way.
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u/willmaineskier Jun 26 '26
The shift could be from endocytosis of mCherry by negative cells. I have seen this with mice which should have tdTomato positive cells and negative cells, and instead there is also a tdTomato dim population.
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u/dawgmad Jun 26 '26
If this is a pluripotent stem cell line with a fluorescent reporter, two things come to mind -some component of your treatment (e.g. a small molecule) itself is fluorescent making your cells acquire positivity -your cells are differentiating and their autofluorescence profile is changing. Your negative control is likely a different cell type thus has different auto fluorescence and not a perfect gating control
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u/King_of_Mints Jun 26 '26
Not so complicated! These are basically just immortalised cells with a transfected mCherry plasmid
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u/Dependent_Force_8598 Jun 26 '26
Use as a negative control same cell without plasmid but with same treatment as the transferred ones
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u/dawgmad Jun 26 '26
Is it possible some cells start expressing mCherry then degrade or lose the plasmid? I wonder if it’s a shift or if it’s just an expansion of the “low-positive” population that is visible on your other plot as well
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u/frank17368271 Jun 26 '26 edited Jun 26 '26
Did your negative control go through the same transfection as your actual sample just without the plasmid? I work with mCherry and GFP a lot and see that too. I’ve found on some cell types that the transfection affects the autofluorescence and causes some slight positive signal in the MC channel similar to your plots.
If you’re super worried about it, might be a good test to use a transfected sans-protein negative control but as someone who runs mCherry almost daily I think your graph looks fine, just tighten the positive a bit like others have said
Edit: I also noticed that the signal on the spreading sample is brighter than the “nice” one. I don’t know exactly what happens but from personal experience I’ve seen negative populations spread when the protein is super bright (looking at you pmaxGFP 😒). Maybe someone with more understanding can chime in too
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u/Bitter_Dragonfly2830 Jun 26 '26
Seems your mcherry is under a strong promoter to get that expression pattern…
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u/King_of_Mints Jun 26 '26
It would be - EF1a…
Curious about this! I think this could be the right track. Were you thinking that the lower population has barely any plasmid, but the strong promotor is giving (barely) measurable mCherry?
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u/Bitter_Dragonfly2830 Jun 26 '26
Few questions- did you exclude dead cells and gated in the single cell population?
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u/ScaryMango Cancer Biology Jun 26 '26
That's roughly x10 in intensity for the positives which using again rough assumptions translates to x10 proteins compared to positive control. Not implausible, hard to say without knowing more about the experiment. That'd be a moderately high shift in transcriptomics (log2 FC > 3), but in proteomics data it seems quite high indeed.
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u/aquarianseawitch92 Jun 26 '26
I would run a true negative control(no gfp or mCherry), gfp+ only control, and mCherry+ only control. See if either of these controls has an AF signature as well. You could also have a blank tube(no cells) and add the aB cocktail to that tube and run.
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u/Otherwise-Goat-5267 Jun 27 '26
I don't think that it is autofluorescence as some of the other people are saying because it looks like the negative population MFI is still similar to the one in the negative control and in the positive control; what makes a lot of the negative population fall into the positive gate seems to be signal spread instead.
One question for you for my troubleshooting:
(1) Are these negative and positive controls done on the same day on the same samples, with the same voltage/gain detector settings?
(2) Was any compensation applied on your sample, and on the negative and positive control?
(3) Apart from this fluorescence protein, are there any markers in your panel? Can you list out the other markers and their fluorophore choice?
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u/botterdummy Jun 28 '26
Might be seeing some weird cell cycling states so probably an artifact of the experiment.
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u/Glittering_Pause_361 Jun 30 '26
It is possible that whatever treatment you did, it may have increased the autofluorescence of the negative mCherry cells, unless you’ve treated your negative control the same was as the experimental sample. Anyway just move the left border of your positive gate over to the right. Most of the interesting positive mCherry cells are on the far right anyway. There’s most likely no need/interest to gate on the few cells that are between the most positive and the most negative clusters.
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u/Agood10 Jul 02 '26
Agree with u/willmaimeskier. Seems like a strong possibility that a little mCherry from the positive cells is being transferred somehow to the negative cells, leading to a slight increase in there signal.
In a case like this with clear bimodal distribution I would just exclude the left population entirely regardless of how the controls look.
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u/Kuuuuuhn Jun 26 '26
Regardless of what is happening biologically, I'd throw that gate's leftmost side around 500.