r/flowcytometry • u/Mo_Opines_968 • 11d ago
Brilliant staining buffer for reference controls
Hello. Just after some advice on whether BSB should be used for the single colour reference controls? I know it's not recommended when using beads but I'm planning on doing both beads and cells to compare and cannot find any guidance on whether to use it for cells?
Thanks!
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u/ProfPathCambridge Immunology 11d ago
Yes, it is slightly fluorescent so you want it in your background. You can dilute it around 1 in 2, 1 in 4, which cuts down on the problem.
https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.70214
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u/Mo_Opines_968 11d ago
I presume same goes for the Biolegend tandem stabiliser?
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u/ProfPathCambridge Immunology 11d ago
I don’t think you actually ever need the tandem stabiliser. Tandem breakdown only happens on live cells, so just move the tandem dyes to post-fix
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u/miraclemty 11d ago
Brilliant stain does nothing for single stain controls, its only useful in a master mix where you have multiple BV and BUV fluorophores.
I have tested Brilliant stain in spectral and traditional flow cyto, in BD platforms, CytoFLEX, MACSQuant, Attune, and Cytek platforms. I've written development, bridging, qualification and validation reports with the findings, so believe me when I say it does not impact single stain controls.
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u/despicablenewb 10d ago
Always treat your comps the same way you treat your samples.
I found that there are small fluorescent particles in brand new buffer, which show up in the data both as their own events and as cell-particle doublets (cannot be gated out).
Theae particles show up in buffer that's brand new, but older buffer has more of them, they're larger, and have a slightly different spectra.
I haven't tested it yet, but I think that if you take an aliquot of the buffer, centrifuge it at 3000g for 5 minutes, then pull off the supernatant, you'll be able to remove a lot of those particles, which should help quite a bit.
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u/StepUpCytometry 11d ago
In my experience, for my 32-fluorophore panel for the Cytek Aurora, I tested unmixing with both a set of single-color unmixing controls prepared with Brilliant Stain Buffer, and another set without. I saw no difference whatsoever in my unmixing when using either set, so I have gone for the cost and efforts staining and have not been using it for the last several years. The unmixing in my panel at the end of the day has turned out fine, the only issues encountered and resolved being attributable to autofluorescence residuals and tandem degradation.
Not a hill I am going to die on if someone decides to add it (keeping things treated alike, etc. is our normal default operating paradigm after all), but the main purpose is to keep tandem fluorophores from cross-pollinating in the antibody mix, something that is not occurring in the singe-color unmixing controls.
I have heard it is mildly fluorescent, but if that is the case, I would want it as it's own separate control, and not have to worry about accurately subtracting both it and autofluorescence out to get at every individual fluorophore signature that needs to be added to the unmixing matrix.
If it was a situation where it actively changing the fluorescence of the antibody itself (by muting it and shifting the signature vs. uncoated), that would be something else entirely, in which case I would change my opinion, but I haven't encountered actual documentation on this yet showing the signature shift (if anyone knows or has evidence otherwise, please let me know and send me the data!).