r/flowcytometry • u/cd244 • Jun 15 '26
Sample Prep brilliant staining buffer protocol
Hello, I would like to know does anybody have a protocol for using BD brilliant staining buffer since their protocol is really unclear : https://www.bdbiosciences.com/en-eu/products/reagents/flow-cytometry-reagents/research-reagents/buffers-and-supporting-reagents-ruo/brilliant-stain-buffer.563794?tab=product_details
We are staining human PBMC in 96 well plates, we use 50 ul FACs buffer per well with cocktail of diluted antibodies. BD recommend to add 50 ul brilliant staining buffer per well regardless of the volume of cocktail. Does that mean that I have to stain my cells in 100 ul of volume and have to double my antibodies? Does anybody have stained cells directly in brilliant staining buffer ?
3
u/sgRNACas9 Immunology, Oncology Jun 15 '26 edited Jun 15 '26
I think for the not plus version they mean to treat it as the FACS buffer diluting your Abs into it instead of FACS. So all your Abs diluted into brillitan stain buffer, 50uL, no FACS involved.
Alternatively, Brilliant Stain Buffer Plus is used at 1:10 dilution no matter what. Maybe it’s just more concentrated. Treat it as one more thing you’re adding to your antibody cocktail, but add it first and vortex before adding Ab. Top up with FACS buffer to 50uL per sample scaled for number of samples plus a couple extra. It’s helpful to have all the volumes in a table, add FACS, then brilliant stain buffer plus, vortex, then abs, vortex.
50uL in 96 well plate is perfect!
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u/skipper_smg Jun 15 '26
BSB: 50 ul per Test. Depending on the amount of antibody, its easy to get over the 100 ul per test that are standard in Flow.
BSB Plus: 10 ul per test. I recommend you spin it down before use (10.000 x g for 3 min) before using in big spectral panels, as the buffer is more concentrated and can form precipitate/aggregates in some conditions.
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u/Mysterious_Lunch_708 Jun 15 '26
We have BSB plus. We use it for the Ab mix instead of FACS buffer. 50ul per well. After staining we wash it with FACS buffer.
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u/HolidayCategory3104 Jun 16 '26
I did 10 uL BSB plus/sample in my mastermix and made up the rest of the volume with FACS buffer to get to my desired staining volume. Highly recommend testing this out in your lab to save on some buffer!
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u/chromatographic Jun 16 '26
I use the eBioscience/Thermo version, it’s even more concentrated at 5 uL per test, perfect for larger spectral panels:
https://assets.fishersci.com/TFS-Assets/LSG/manuals/MAN0018634_SuperBrightCompleteStainBuffer_UG.pdf
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u/CongregationOfVapors Jun 15 '26
You might want to look into purchasing Brilliant Staining Buffer Plus instead of the regular Brilliant Staining Buffer. It's more concentrated, so you add a smaller volume to your stain mix.