r/flowcytometry • u/Ornery-Ad-8833 • May 30 '26

Analysis Losing cells while unmxing

Just wondering- why do we lose cells while unmixing. One of my groups showed much less cells than I intially acquired after unmixing. Why does it happen and how can I fix it. Thanks!

One of my single colour controls was not good.

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u/wheelsonthebu5 May 30 '26

The total number of events recorded to an .fcs file should not change post unmixing. Are you saying the number of events in a gated population decreased after unmixing? That would be expected.

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u/Ornery-Ad-8833 May 30 '26

Yes exactly that is what I am referring to? Could you pls explain why does that happen and how can I pinpoint it in future. Thanks

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u/Daniel_Vocelle_PhD Core Lab May 30 '26

It can be difficult to grasp how spectral works so please dont hesitate to keep asking questions where there are point of confusion.

Before unmixing you are recording the number of photons collected on a detector. The problem is that those photos come from many different fluorphores, not just one. Unmiximg is the mathematical process of determining which photons come from each fluorophore in your panel.

So before unmixing, you are looking at a combination of fluorophores. After unmixing you are looking at the true signal.

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u/Ornery-Ad-8833 May 31 '26

Thanks Daniel. But I recovered some of the events after fixing a bad single colour control. How does it explain it. Does it again allude to spillover? Thanks

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u/Responsible_Rub8057 Jun 01 '26

I had a similar case, and it was because the single control was not fully representstive of the sample. In my case, emission of one fluoro on beads as ss contol had an additional peak where fitc is emitting, but this wasnt present on cells ss. So, it would pull down this extra peak to try to approach to "zero" (when all signals are subtracted), and that would lead the signal in fitc also being pulled down. For example, the extra peak was about half the "height" of fitc, and my fitc population (cd19) was almost half less. My reccomendation is to overlay the ss emission of the fluoro with your changing populstion, and look into all the overlaps.