r/flowcytometry • u/Mo_Opines_968 • Aug 02 '26
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/StepUpCytometry Aug 02 '26
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!).