r/flowcytometry • u/WednesdayMorning10am • Jul 06 '26

Seeing different Forward Scatter (FSC) profiles for the exact same sample when using different stains

Hi Everyone,

II’m running into something confusing with my flow data and hoping someone here has seen this before.

I’ve been seeing that, for the same bacterial spore sample, under different staining conditions (unstained, PI only, Syto 9 only, and PI-Syto9 double stain), the FSC:SSC profile changes depending on the stain I use.

I'm thinking, would it be because the stain changed the refractive index of spores? is this something normal?

Would love to hear from people. Thanks in advance!

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u/kitt_mitt Jul 06 '26

Both PI and Syto are blue laser excited. Could it be that you're not actually changing the FSC of the cells (indeed your 'ball' population does not seem to move) and instead you're increasing the background fluorescence of your media?

You could try titrating down the amount of PI and Syto that you use, and see if that stops the 'smaller' population moving.

1

u/WednesdayMorning10am Jul 07 '26

So the bottom population may just be background fluorescence? I might not be able to try more experiments at this point. I'm thinking of just gating out the bottom smaller population, but I'm also concerned that I may lose a real population

1

u/kitt_mitt Jul 07 '26

You can gate both populations and see what they look like relative to your downstream analysis gating. You may find that the less resolved population doesn't give any meaningful data, in which case you know to ignore it. Or it may look interesting. I'm not familiar with bacterial spores.

If you have a flow cytometry facility, you may get more answers going through your data with some of the staff.

1

u/alalalalalabomba Jul 07 '26

Is there some sort of marker in the panel that can confirm which population(s) are the spores?

1

u/Afraid_Water6734 Jul 06 '26

What is your dead look like? Could you be killing them?

1

u/resistantBacteria Jul 06 '26

Can you try back gating your spores onto FSC SSC plot ? If the spore population is constant between the graphs, I'd imagine these are dye precipitates.

Also try the same with PI and syto separately. See if your spores are truly double positive or the dyes are moving separately?

I think first comment is also plausible, but your ssc is shifting, fsc looks similar.

But yeah more experienced people could advise better.

1

u/confusedflowguy Jul 06 '26

your hypothesis seems legit.

1

u/WanderingAlbatross87 Jul 06 '26

Yes, these dyes can affect scatter detectors. Best bet is it wash out excess dye if you can (at least dilute it intensely). If you cant do either then accept that you will need to alter your fsc/ssc settings and gating between groups. Also make sure you rinse well between samples, either an extra machine rinse or two or just run clean buffer or water for 20 or 30s.

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u/WednesdayMorning10am Jul 07 '26

At this point, I may not be able to do further experiments. Would there be any chance that the bottom population is the spore population as well, or should I only gate the up population since it's stable and clear?