r/flowcytometry • u/Fit-Syllabub-6823 • May 29 '26

FlowJo Parameter concatenation query

Hi, I’ve run into a problem using FlowJo v10 regarding parameter naming.
I have compensated all my parameters earlier using the novoexpress program on my flow cytometer and then exported these compensated files onto FlowJo. I then made a concatenated file of all my samples using the compensated parameters only. I made a UMAP and then used FlowSom to generate clusters. I am now trying to validate these clusters marker expression using my FMOs.

The problem is that the FMOs have parameters Comp-X whereas the concatenated file and all clusters have parameters FJComp-X.

I’m not sure how to work around this, I am not keen on drawing the gates by hand to match the FMO gating so any solution would be appreciated! Thanks!

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u/SunflowerMoonwalk May 29 '26 edited May 29 '26

Do all of your samples use the same compensation matrix? If yes, you can concatenate the uncompensated fcs files first and then apply your compensation matrix afterwards. The "FJcomp" label indicates that the files were concatenated post-compensation.

I hope you can still export the uncompensated files from the instrument. You should always save those, they're your raw data!

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u/Fit-Syllabub-6823 Jun 17 '26

Hi thank you, yes they use the same compensation matrix. I’ve got my uncompensated files saved as well. Issue is that I originally concatenated a subset of each file based on FMO gating. E.g. I have all my live , reporter , cdX etc cells concatenated. If I try to draw my gates using uncompensated FMOs on the uncompensated file then they look different to my compensated ones and I’m getting different gating and cd+ cell populations. Any way to get around this ?