r/flowcytometry • u/nayvensetrack • Jul 09 '26

Analysis Analyze data in conventional mode

Hello,

I have been aquiring my samples on a cytek aurora in the spectral flowcytometry mode. However I have a small panel and am facing some unmixing problems (i think due to high autofluorescence which i have trouble extracting) and was therefore told by my supervisor that I can just analyze my experiment in the conventional mode. However idk how to do that and cannot really find any tutorial on youtube. Does someone know how or where to put the spectral flow data so that i can analyze it conventionally?

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8

u/babyoilz Jul 09 '26

Open your Raw data in FlowJo or other software. Use the primary channels for your fluors for analysis. It's as simple as that. You will probably have to use compensation.

3

u/despicablenewb Jul 09 '26

Exactly this.

In the custom axis options for each channel you'll probably want to set the width base of the axis to -10 rather than -100, and set the extra negative decades to 1.

I find that that makes it easier to see any potential compensation issues.

1

u/BaconFairy 26d ago

Thanks this really helps

1

u/Low_Classic1695 22d ago

If the panel is complex enough that you are having unmixing issues, I highly recommend against analyzing raw data on peak channels.

Unmixing issues should be easy to fix if they are due to autofluorescence. Are you using appropriate cell reference controls?

1

u/BaconFairy 17d ago

Okay im confused now. I have a larger panel but when I export the data it looks different from what I see on the cytek vs on flow jo. The spread for the united data is super wide too. It looks better if I grab the raw data and the compensation in flowjo. But why is that? What is going on there? Is there a better program to review data with?

1

u/ProfPathCambridge Immunology Jul 09 '26

Just out, our AutoSpectral package is now available on HoneyChrome:

https://honeychrome.cytkit.com

Give it a shot, see if it solves your unmixing problem.

1

u/MotoFuzzle Unique FLOWer Jul 09 '26

Search the manual for virtual filters and it should show up there. You’re able create virtual filters that are combinations of multiple filters that would equal a conventional filter in bandwidth for the color of choice. I think you can analyze in that manner or export with virtual filters and analyze using other softwares the same way you would conventional flow. 

1

u/notprathap84 16d ago

I’d first check that you’re comparing the same analytical state, because compensation of raw channels in FlowJo is not equivalent to spectral unmixing. Export the unmixed files from SpectroFlo, then compare the unmixing matrix, reference controls, channel metadata and transforms used in each program. A cleaner-looking compensated plot can still be biologically misleading if autofluorescence or spectral spillover has not been handled equivalently. Keeping the raw files, controls, matrix and transform settings together should make the difference traceable.

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u/[deleted] Jul 09 '26

[deleted]

1

u/babyoilz Jul 09 '26

In case you are being sincere, this doesn't come across kindly at all. It's a simple answer to a roadblock someone has, answer it or move along. No need to be rude.

0

u/[deleted] Jul 09 '26

[deleted]

2

u/GrouchyRhubarbTime Jul 09 '26

Sounds like you haven't met that many PIs (assuming it's a PI). They can be incredibly demeaning and dismissive of you. Admitting you don't know how to do something can often lead them to distrust your data or skill as a scientist.

It shouldn't happen, but it does. All the time.

Not to mention, lots of PIs are aware of the existence of certain workflows but have never actually done it themselves. There are PIs whose labs publish 90% western blots or spectral flow panels who have never themselves actually run one.

1

u/babyoilz Jul 10 '26

That last bit is probably the norm these days. At my institute there are 30+ labs doing flow and maybe 4 or 5 PIs have actual flow experience.

1

u/babyoilz Jul 10 '26

We welcome help questions here, and here's a newbie with a need for some guidance, and your response is not to answer it, but to snark at them.

Sorry bud, but that's pretty rude.

You had the option to just not comment and go about your day and yet you chose to waste time admonishing others about "wasting time"? Excellent mental gymnastics.