I performed spectral flow cytometry on a Cytek Northern Lights, but a few samples appear to have taken up bubbles during acquisition. I later realised some wells likely had <100 µL volume due to uncalibrated pipettes, so I suspect the instability is from low volume/bubbles during uptake.
The issue is that the affected samples/reference controls contain obvious unstable acquisition periods that I want to remove before unmixing.
What I’ve tried so far:
In SpectroFlo, I can create a time gate (P2) before unmixing, but during unmixing, it still seems to reference the original ungated events rather than the P2 time-gated population. I also can’t seem to change the channel/source population used for unmixing.
I then exported the raw FCS files into FlowJo, applied time gating there to remove the unstable regions, and exported the gated populations as new FCS3 files with all uncompensated parameters.
However, when I try to re-import these gated FCS files back into SpectroFlo for unmixing, I get “Possible file corruption or wrong files uploaded”.
I suspect this may be due to SpectroFlo/Cytek metadata being altered during FlowJo export, even though the files open fine in FlowJo.
Has anyone dealt with this workflow before on Cytek instruments?
Is there a proper way to time gate/remove bubbles BEFORE unmixing in SpectroFlo?
Can SpectroFlo unmix from a gated population rather than the parent acquisition?
Is there a known method for exporting FlowJo-gated Cytek files back into SpectroFlo without triggering file corruption errors?
Any advice would be massively appreciated 🙏🏼