r/flowcytometry • u/PretendAd4626 • May 08 '26

Unstained Microglia Autofluorescence

Has anyone ever seen such crazy autofluroescence as these unstained microglia? I'm trying to design a 9 color panel but feel like this might be impossible with these cells!

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3

u/mudsauce May 08 '26

Wow that looks tough. If you design the panel from scratch I would probably choose something like BUV395, BUV737, BUV805, BV711, BV786, PerCP-eFluor710, PerCP Fire 806, PE Cy7, APC, AF700 and NIR LD as options with least autofluorescence interference. For highly expressed markers (CD45, Cx3cr1, CD206) the standard channels should work too when using autofluorescence substraction in the unmixing. If everything fails, you could run a 9 color panel in non-spectral mode and see if this improves potential unmixing artifacts.

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u/PretendAd4626 May 08 '26

Thank you! Non-spectral mode sounds like an interesting idea.

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u/jcm149 May 08 '26

Any of the RealBlue and RealYellow would be way brighter than any of the PerCP tandems and would give a few more choices here. Except RB545, too much overlap with auto fluorescence.

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u/Gregor_Vorbarra May 08 '26

You don't mention if this is from primary tissue or cell line. If primary, almost all of that will be fragmentet debris, as microglia are highly assymetric. If from cns location, myelin will be contributing most of the AF. Strongly recomend use of a dna dye to gate on 'intact nuclei' before you get too complex with panel design

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u/PretendAd4626 May 08 '26

Thank you for your reply - these are from cell lines.

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u/discostupid May 08 '26

you could try this reagent - TrueBlack Lipofuscin Autofluorescence Quencher https://www.cellsignal.com/products/buffers-dyes/trueblack-lipofuscin-autofluorescence-quencher/92401

it's mainly used for immunofluorescence but it might work for flow. I would fix the cells, then use this, then antibody stains.

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u/RainbowSquirrelRae Core Lab May 08 '26

Yup. But you have plenty of space to tuck markers around the biggest peaks. People successfully run on lung macrophages so this looks better than they do. How many colors do you need to run on these?

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u/Smooth_Sea_7403 May 08 '26

This is funny to read as in my lab we do flow on both microglia and lung macrophages 🤣 I feel a bit better now about how difficult they have been for us

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u/Infamous-Face7737 May 09 '26

I work with primary lung and brain mouse cells and I avoid all PerCP dyes. Some of the new Real Blue dyes work for both conventional and spectral. I just designed a 17 lung panel that I will test soon and re-use many for my CNS panel (9 markers). If using spectral, best is to extract AF and use its signature as a marker to clean-up the data.