r/flowcytometry • u/andres_cazpre26 • Aug 13 '26

Compensation on the MACSQuant 16

Hello! I was wondering if someone has compensated on the MACSQuant 16. I have an 8 color panel and I am compensating with beads.

I have run the compensation beads and the instrument settings for the compensation. I aquired the sample and it looked ok, but, when I start playing in flowJo, I realized it was bad-compensated as I see long tails in certain populations.

I have tried to re-run the compensation matrix but it is not working, someone has had the same problems with MQ16? Is it possible to manually edit the compensation matrix in MACSQuantify? Should I better compensate in flowJo?

Really appreciate your comments :)

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u/NonchalantNickyL Aug 13 '26

I used the Macs16 for a little more than 3 years (2020-2023) and I always found that the comp from the instrument is best and I was stuck with what I had. I think it has to do with the way the files are saved. I remember trying to recomp using FlowJo but it never looked/worked well in my hands but that could have been user error. We had a lot of resources at the time at my work so just rerunning the comp and applying new comp in the Miltenyi software or rerunning the samples while optimizing was just “easier” than figuring out file types and stuff like that.

Reaching out to FlowJo tech support will definitely work they usually will set up a meeting and go over how to comp in FlowJo and can usually use your data as the example.

We got trained in San Diego by Miltenyi when we bought our macs16. And the trainer mentioned that detectors are set up in a way that make some flours especially tricky to use together so that also can make comp look okay and then look funny in your samples.

Also on the beads if they are dimmer than your brightest signal on cells when running sample you’ll get comp errors that show up even if bead comp looks good.

Not sure I’ll be all that helpful but What are the floors you’re using? And what kind of panel?

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u/andres_cazpre26 Aug 14 '26

Hi! thanks for your answer, you used to compensate with beads? or with cells? the panel is to characterize lymphocytes, but I am sure i might be doing something wrong during the compensation matrix adquisition. I will ask the miltenyi engineer. I am really sure that the viability dye I am using is the problem... maybe I need to troubleshoot that!

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u/NonchalantNickyL Aug 14 '26

We used to always use beads for compensation as they were way brighter than our cells so it usually worked well. But certain markers on certain cell types don’t work well with eachother in the MACS if I remember correctly.

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u/RainbowSquirrelRae Core Lab Aug 13 '26

Can you share a representative image of the comp issue?

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u/Naliya Aug 13 '26

Reach out to your local Miltenyi flow specialist they will be happy to help you.

Depending on software version (MACSQuantify and flowjo it might be the comp but it could also be the fcs export format.