The issue
Interactions between Brilliant dyes on antibody conjugates. Creates compensation-like artefacts where cells staining with one Brilliant-conjugated antibody are artificially positive for others.
The reagent
The appropriately named Brilliant Stain buffer.
Available from BD and ThermoFisher:
Also available in a more concentrated format:
What is it?
Probably monomers of the base SIRIGEN dye, according to Florian Mair. The spectrum of the BS buffer resembles that of Super Bright 436, as we'll see later.
Alternatives
None, just the variants above. Using less antibody limits the artefacts.
What it’s supposed to do
Reduce dye-mediated interactions between Brilliant Violet, Brilliant Ultraviolet, Brilliant Blue and Super Bright conjugated antibodies.
Brilliant Stain Buffer also helps reduce background in human whole blood assays because it contains polyethylene glycol (PEG). A lot of fluorophores contain PEG, so the presence of PEG in the buffer swamps interactions between serum antibodies and the fluorophores. Why do people have anti-PEG antibodies? Well, there's PEG in vaccines, too, and a lot of people got vaccinated recently. Read more here.
Examples
You've probably all seen BD's classic examples using CD4 and CD8 to fix compensation-like artefacts with Brilliant Stain buffer. A lot of the time, the effects are more subtle. In the example below, there's a correlation between ICOS and CD25 on CD4 T cells; some of that is biologically correct, and some of it goes away when we use the Brilliant Stain buffer.
With mouse cells, we tend to use lower concentrations of antibody, and it can be hard to see much benefit of the BS buffer (see below). Not all Brilliant conjugates have this issue, and some that do have it so badly that it cannot be fixed with the buffer. I'm not sure why this is, but my guess would be that some antibodies have more unconjugated fluorophore in the vial, which would be more likely to cause this.
Any detrimental effects?
Yes. Moderate effects that can be dealt with, mitigated, and are usually worth it.
The Brilliant Stain buffer adds background fluorescence to compensation beads, so don’t use it with beads. BioLegend maintains that this isn't a problem with their beads.
Given that it adds fluorescence to beads, we can expect this to happen with cells too:
The black spectral curves above are from human PBMCs stained with either a Super Bright 436 conjugate (top panel), or the recommended 50ul of Brilliant Stain buffer (from ThermoFisher, lower panel). You can ignore the red curves--those are benchmark spectral traces for BV421 and Super Bright 436 that were recorded last year and have been updated since by the Cytek Assay Settings. Note that this divergence from the correct spectra occurs with all reference controls over time and gives the lie to the idea that you can unmix accurately with stored controls (something for a future discussion).
This is likely to be more of an issue with prolonged intracellular staining, as I'm showing here. You should expect some effect along these lines with any exposure to BS buffer, perhaps particularly in phagocytic cells like monocytes.
In this example, we can see that adding Brilliant Stain buffer increases the background in the Super Bright 436 channel.
In some cases, using a lot of BS buffer can reduce cellular viability. Here's an example of that with cryopreserved PBMCs. The buffer contains a low level of PEG, which explains why it looks a bit filmy, and PEG can reduce cell viability.
How much to use?
As little as you can get away with. The amount you need varies in proportion to the concentration of Brilliant dyes you’re using. The recommended test is 50µl. You’ll probably only need that for human cells stained with the recommended concentrations of antibody.
As we showed in this paper, by reducing the antibody concentration down with overnight staining, these non-specific interactions are greatly reduced. For overnight staining, I generally only use 10µl or less. In some cases, with this approach, it is unnecessary.
When do you really need it?
Whenever you are using more than one Brilliant or Super Bright dye at the same time. Also if you're working with human whole blood due to the prevalence of anti-PEG antibodies.
Don’t use it with compensation beads. You don’t have more than one Brilliant conjugate in this scenario. See Laura Ferrer-Font’s paper. Panel Optimization for High‐Dimensional Immunophenotyping Assays Using Full‐Spectrum Flow Cytometry - Ferrer‐Font - 2021 - Current Protocols - Wiley Online Library
Bottom line/how to get the best results
Test how much you really need in your assay. For this, identify any staining pattern that looks odd and test it with an FMO control. Are you getting a correlated expression pattern with both Brilliant dyes present? If so, titrate in the buffer until the effect goes away. My rule of thumb is to use slightly more than needed to provide a bit of a safeguard.
Reagents used: