u/Zestyclose_Zebra6783

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/Zestyclose_Zebra6783 — 12 days ago