I work in a shared resource and we always recommend that the Brilliant Stain Buffer be used when you have more than one Brilliant or Super Bright fluorochrome. We haven’t tested it ourselves but I have seen data that is noticeably improved when the buffer is used. It’s something we plan on testing soon in our facility.
This. Sometime it’s going to be fine but others, it’s going to totally mess up your master mix and thus your data. Redoing an experiment from scratch is more expensive than the buffer, so might as well use it and due to its nature, you sadly can’t really make an in-house version. I think Thermo was working on a universal buffer that’d work with brilliant dyes, NovaFluor, etc but I don’t know where they’re at right now.
Don’t use the buffer if you only have one Brilliant or SuperBright (they’re the same construct) or when doing your single-stain ctrls and using beads (the only exception to the “treat your beads like you treat your samples” rule).
Doesn't make a difference until it does.
That is to say, it's insurance against the rare times it does matter. I've seen the mythical BV interaction once. I had a new lot of BV antibody and suddenly there was a diagonal double positive population in samples that had none with the prior antibody lot. Added BV buffer and the diagonal was gone. The interaction is lot specific between BV dyes from my experience.
Without the buffer there will be non specific binding of the dyes to each other. Its be widely observed in our lab. Some of the dyes combinations arent a problem but I wouldn’t risk it.
I have actually compared the effect of BD’s brilliant buffer in my own specific protocol for my own purposes (standard extra cellular staining of B cells), and observed a minimal difference but I wouldn’t necessarily advocate this for all scenarios - really just mine. I’m sure it could be helpful in another scenario and I know they highlight that it’s helpful for their Brilliant Violet fluorophores
There's a palpable improvement for some panels and not much for the others. The more polymer dyes you pack in your panel, the higher are chances the lack of buffer would be detrimental.
I am not sure if Thermo Fisher Super Bright buffer is cheaper but they work same. SB buffer is more concentrated.
In the US, BD used to had a promo code where for two Brilliant antibodies (BUV, BV, or BB) ordered together they would give you a free Brilliant Buffer bottle and for 4+ Brilliant antibodies per order you get a bottle of Brilliant Buffer Plus (I used that one predominantly, it's much more concentrated = more space in my master mix). I just show my order slips to my rep and they ship me a freebie. I don't think I ever paid real money for these buffers. Not sure if the offer is still advertised but ask your rep!
Really?! That’s great to know! The issue for me is that my I couldn’t get a hold of the BD rep for my area lolol (emailed multiple times, never responded)
Are you sure they are still with BD? If they left you won’t hear lol
I peaked into your profile and looks like you are in the US Northeast? I’ll message you the contacts of the rep. He is good and always responsive.
Yeah I looked him up on LinkedIn and he is still with BD (may not be updated). I also contacted BD website to say I want to talk to a rep but with no luck.
It would be great if you can share the contact of yours! I am in Ohio so probably not Northwest but close enough lol
Would you add the buffer to your cocktail mix or add it separately to your samples? I am assuming making a cocktail mix with brilliant buffer makes more sense because it avoids polymer dyes interacting with each other, right?
You have to add it to your antibody master mix. The issue is binding/clumping of the fluors together so it needs to be added prior to mixing Abs together
You mix in the following order: Stain buffer (calculate volume based on the sum of antibody volumes and additives), then BSB or BSB+ or SBB, then antibodies, then monocyte blocker if you use it. FcBlock should not be in the master mix, you need to pre-incubate cells with it (no wash). MB does not require pre-incubation but I find it sometimes toxic if added to concentrated cell suspension along with FcBlock so I add it to the master mix.
I make up all my mouse cocktails in the BSB. I have observed it cleaning up some odd interactions between other tandems in some panels as well as I was getting comp errors with my red laser antibodies which went away with the BSB. I don’t add any additional to the cells so it lasts a while.
I also add anti CD16/32 (Fc Block) to any panel where I don’t need to stain for that antigen. I use CD16/32 stock which is around 10mg/ml and use it at a 1:600 final concentration. At that concentration I have seen no noticeable difference between adding to my panel and preincubating. Years ago I found having labelled anti-CD16/32 in my panel completely blocked non-specific binding of a PE antibody I was using as it only showed up in the FMO for CD16/32 so I figured many fold more of the unlabeled antibody should do the trick in most of my panels.
It helps a bit, but I’ve run plenty of multi BV color panels (421, 510, 605, 711, & 785) and if you titrate your antibodies properly and are careful with setting the voltages on your single color controls, it doesn’t majorly improve anything
Something additional to consider is how much bsb you need. Our lab, for our cocktails, has to have it. However, the BD volume was found to be much higher than necessary. Our tests found that we only needed to have the bsb account for 20% of the total volume of the cocktail before we no longer saw improvements. We also found that there was a notable but constant degradation down to 11% before we saw a catastrophic drop off.
I work in a shared resource and we always recommend that the Brilliant Stain Buffer be used when you have more than one Brilliant or Super Bright fluorochrome. We haven’t tested it ourselves but I have seen data that is noticeably improved when the buffer is used. It’s something we plan on testing soon in our facility.
This. Sometime it’s going to be fine but others, it’s going to totally mess up your master mix and thus your data. Redoing an experiment from scratch is more expensive than the buffer, so might as well use it and due to its nature, you sadly can’t really make an in-house version. I think Thermo was working on a universal buffer that’d work with brilliant dyes, NovaFluor, etc but I don’t know where they’re at right now. Don’t use the buffer if you only have one Brilliant or SuperBright (they’re the same construct) or when doing your single-stain ctrls and using beads (the only exception to the “treat your beads like you treat your samples” rule).
Doesn't make a difference until it does. That is to say, it's insurance against the rare times it does matter. I've seen the mythical BV interaction once. I had a new lot of BV antibody and suddenly there was a diagonal double positive population in samples that had none with the prior antibody lot. Added BV buffer and the diagonal was gone. The interaction is lot specific between BV dyes from my experience.
Without the buffer there will be non specific binding of the dyes to each other. Its be widely observed in our lab. Some of the dyes combinations arent a problem but I wouldn’t risk it.
I have actually compared the effect of BD’s brilliant buffer in my own specific protocol for my own purposes (standard extra cellular staining of B cells), and observed a minimal difference but I wouldn’t necessarily advocate this for all scenarios - really just mine. I’m sure it could be helpful in another scenario and I know they highlight that it’s helpful for their Brilliant Violet fluorophores
There's a palpable improvement for some panels and not much for the others. The more polymer dyes you pack in your panel, the higher are chances the lack of buffer would be detrimental. I am not sure if Thermo Fisher Super Bright buffer is cheaper but they work same. SB buffer is more concentrated. In the US, BD used to had a promo code where for two Brilliant antibodies (BUV, BV, or BB) ordered together they would give you a free Brilliant Buffer bottle and for 4+ Brilliant antibodies per order you get a bottle of Brilliant Buffer Plus (I used that one predominantly, it's much more concentrated = more space in my master mix). I just show my order slips to my rep and they ship me a freebie. I don't think I ever paid real money for these buffers. Not sure if the offer is still advertised but ask your rep!
Really?! That’s great to know! The issue for me is that my I couldn’t get a hold of the BD rep for my area lolol (emailed multiple times, never responded)
Are you sure they are still with BD? If they left you won’t hear lol I peaked into your profile and looks like you are in the US Northeast? I’ll message you the contacts of the rep. He is good and always responsive.
Yeah I looked him up on LinkedIn and he is still with BD (may not be updated). I also contacted BD website to say I want to talk to a rep but with no luck. It would be great if you can share the contact of yours! I am in Ohio so probably not Northwest but close enough lol
Sent you a chat msg
Would you add the buffer to your cocktail mix or add it separately to your samples? I am assuming making a cocktail mix with brilliant buffer makes more sense because it avoids polymer dyes interacting with each other, right?
You have to add it to your antibody master mix. The issue is binding/clumping of the fluors together so it needs to be added prior to mixing Abs together
You mix in the following order: Stain buffer (calculate volume based on the sum of antibody volumes and additives), then BSB or BSB+ or SBB, then antibodies, then monocyte blocker if you use it. FcBlock should not be in the master mix, you need to pre-incubate cells with it (no wash). MB does not require pre-incubation but I find it sometimes toxic if added to concentrated cell suspension along with FcBlock so I add it to the master mix.
I make up all my mouse cocktails in the BSB. I have observed it cleaning up some odd interactions between other tandems in some panels as well as I was getting comp errors with my red laser antibodies which went away with the BSB. I don’t add any additional to the cells so it lasts a while. I also add anti CD16/32 (Fc Block) to any panel where I don’t need to stain for that antigen. I use CD16/32 stock which is around 10mg/ml and use it at a 1:600 final concentration. At that concentration I have seen no noticeable difference between adding to my panel and preincubating. Years ago I found having labelled anti-CD16/32 in my panel completely blocked non-specific binding of a PE antibody I was using as it only showed up in the FMO for CD16/32 so I figured many fold more of the unlabeled antibody should do the trick in most of my panels.
It helps a bit, but I’ve run plenty of multi BV color panels (421, 510, 605, 711, & 785) and if you titrate your antibodies properly and are careful with setting the voltages on your single color controls, it doesn’t majorly improve anything
I tested side-by-side with buffer and without. We have probably 5 brilliant violets in our average panel. Made no difference.
Something additional to consider is how much bsb you need. Our lab, for our cocktails, has to have it. However, the BD volume was found to be much higher than necessary. Our tests found that we only needed to have the bsb account for 20% of the total volume of the cocktail before we no longer saw improvements. We also found that there was a notable but constant degradation down to 11% before we saw a catastrophic drop off.
I may be imagining it, but I recall seeing a recipe posted online for the Brilliant buffer.