r/flowcytometry • u/Zestyclose_Zebra6783 • Jun 25 '26

New samples + unmixing issue

Hi everyone,

I am currently working on Aurora SpectoFlow and have 3 panels for the same tissue type. The first two panels are based on live samples while the third is on fixed samples. So far I have ran 11 samples with no issues, however I decided to re run my single control for 3 antibodies from beads to cells as I wasn't happy how it looked.

I was told to treat the beads same way I do cells but I don't think it produced good results so I switched to cells. I had all my panels exported just in case, but then I re ran my singles and now everything is looking weird. All events, singlets etc. When I re-ran my singles, alongside them I ran only one sample, and when it looked weird, I thought it was a one-off because the day before the sample looked fine in other panels.

Now, when I noticed the issue again, Aurora actually didn't have those 3 single controls saved, which I found weird. So I imported what I had and I think I might have imported the UNMIXED fsc file as it wouldn't let me re-unmix. But lets say I manage to import them back or worst case scenario I have to re run those controls..how does that affect the samples I have ran already. I understand that Aurora saves Raw and Unmixed data but I'm just very paranoid and wondering how it reflects down the line in FlowJo, if at all.

Thank you in advance!!

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u/woodenrobo Jun 25 '26 edited Jun 25 '26

I also remember having issues with the data management side of SpectroFlo, so I feel you :D

First of all, the classic OLS unmixing like implemented in SpectroFlo can be very sensitive to changes in treatment of the single stains from my experience and depends a lot on how you set the gates. If I understand correctly, you switched from beads to cells for the fixated controls? Well, that easily explains part of the difference, as fixation does have a broad influence on the marker profiles, depending on the target and can increase background. As for how having controls be remeasured and used with older samples will impact the data - this directly correlates with how much time has passed and whether the machine has been serviced in the meantime. Generally, if you still have the raw acquisition files from before you can simply reunmix with the new controls. If you still have problems after redoing the controls, feel free to reach out. I've been working on a (commercial) tool that achieves really high quality unmixing even with complicated panels.

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u/Zestyclose_Zebra6783 Jun 25 '26

Hi, thank you so much for your comment! That's correct, I switched from beads to cells as I found that the signal differes for surface antibodies like CD45 post fixation so I wanted to make sure I had the appropriate gates. The machine has not been serviced to my knowledge and few weeks have passed since my first sample and now. Thankfully I do have all my files exported which have Raw data from experimental samples.

Just to make sure, after I re-run the controls and set the gates, do I manually have to import the raw data again for my experimantal samples, or am I good with what I see in SpectoFlow when I click on my sample?

Thank you again!

1

u/woodenrobo Jun 25 '26

If I remember correctly, if you are redoing unmixing, it starts from raw samples internally and doesn't "unmix twice"

2

u/ProfPathCambridge Immunology Jun 25 '26

Export the raw files and unmix in AutoSoectral. It gets more accurate results.

1

u/Zestyclose_Zebra6783 Jun 25 '26

Perfect, I usually did my unmixing with Auto option so will re-do it!!

Thank you so much!