BD Horizon Brilliant™ BV421 Dyes

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A Bright Fluorochrome Suitable for Low Antigen Density Markers

As one of the brightest fluorochromes available today, BD Horizon Brilliant Violet^™ 421 (BV421) Dye delivers exceptional signal with low spillover. Driven by BD innovation, BV421 is designed to be excited by the violet laser (405-nm) and is best detected using an optical filter centered near 420 nm.

Ideal for conventional flow cytometry and spectral flow cytometry
Suitable in multicolor applications to resolve dim populations
Can be detected by the common 450/50-nm bandpass filter

The BV421 spectrum

Format	Ex _Max	Em _Max	Spectral	Conventional	Relative Brightness	*Spillover (1 = low, 4 = high)**	Alternative To
BV421	407 nm	423 nm	Yes	Yes		1	BD Horizon^™ V450, Alexa Fluor^® 405, VioBlue®, eFluor^® 450, Pacific Blue^™

*Value may vary based on instrument configuration and settings. Spillover ranking is based on cross-laser excitation on five-laser spectral instruments and does not take into account spillover into adjacent detectors.

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Performance

BD Horizon™ BV421 Dye Provides Exceptional Resolution

BV421 provides greater fluorescence intensity and can be used to detect low expression markers compared to Pacific Blue™ and BD Horizon™ V450.

Human PBMC were stained with CD4 (SK3) BV421, V450 or Pacific Blue™. Cells were acquired on a BD FACSLyric Flow Cytometer and analyzed with FlowJo Software.

Applications

BD Horizon Brilliant™ BV421 supports detection across a range of marker expression levels

BV421 is a bright fluorochrome to support detection of markers across a range of antigen densities. Below are examples of BV421 detecting low (PD-1), medium (CD19), and high (CD45RA) expression markers. For further information about pairing fluorochromes and antigens, please reference the Fluorochrome Performance Guide.

RV828 Reagents Lower Expression Surface Marker

Human whole blood was stained with either PD-1 BV421 (EH12.1), CD19 BV421 (HIB19) or CD45RA BV421 (HI100). Samples were processed with BD FACS™ Lysing solution, acquired on a BD FACS Discover™ A8 flow cytometer and analyzed with FlowJo Software.

Intracellular Flow Cytometry

BD Horizon^™ BV421 supports intracellular flow cytometry applications

IL-22 expression in stimulated human PBMCs.

Human PBMCs were stimulated with PMA/ionomycin in the presence of GolgiPlug™ for 6 hours, fixed, permeabilized, and stained with CD4 BUV395 (RPA-T4) and IL-22 BV421 (MH22B2). Representative pseudocolor density plots show IL-22 versus CD4 expression in gated lymphocytes, analyzed using a BD LSRFortessa™ X-20 Cell Analyzer and FlowJo™ software.

Multicolor Flow Cytometry

BD Horizon^™BV421 supports multicolor flow cytometry

BV421 conjugated antibodies can resolve lower expression markers such as human CD127 in a multicolor flow cytometry panel

Reagents Resolution in 24-Color T Cell Activation Panel

The BD Horizon^™ Human T Cell Backbone Panel was complemented with surface and intracellular markers conventionally used to identify human Treg subsets, BV421 CD127 (HIL-7R-M21, 562436), RY586 CD25 (2A3, 568124), AF488 FoxP3 (236A/E7, 561181) and APC HLA-DR (G46-6, 559866). Clear resolution of all the subsets of interest was observed on fixed and permeabilized PMBCs from n = 3 healthy donors. Samples were acquired on a 4-laser BD FACSymphonyTM A1 Cell Analyzer and analyzed with FlowJoTM Software.

FAQs

What is the optimal filter for BV421 on my conventional cytometer?
BV421 can be used with the commonly used 450/50 bandpass filter, but a filter centered closer to the peak emission like a 431/28 bandpass filter would capture optimal signal.
When it comes to BV421 compensation and spillover, what should I consider?
Because BV421 is extremely bright, spread from spectral overlap can be more apparent. This is particularly the case if you are also trying to resolve dim markers in neighboring channels. BV421 works well with either BV480 or BV510. Both BV480 and BV510 will have more adjacent spillover into the wider 450/50 BV421 filter than the 431/28 filter.
If I use BV421 during the intracellular staining step of my protocol, do I need to add Brilliant Stain Buffer (BSB) to both my surface stain and my intracellular staining cocktail?
Yes, it is advised to use BSB if two BVs are used even if the two BVs are stained at different steps. If volume becomes an issue, the more concentrated version Brilliant Stain Buffer Plus is a good option.
What is the difference between BV421 and V450?
There are two main differences between BV421 and V450. First BV421 is a very bright fluorochrome where V450 is classified as Dim. This means you would not assign the dyes to markers in the same way. BV421 should be used for low expression markers, variable expression markers or markers expressed over a continuum. V450 is a good format for high density antigens such as lineage markers. The other notable difference is their emission profiles. V450 has a longer tail with more spectral overlap with BV480 and BV510. This may be more impactful in spectral flow cytometry where the whole spectrum is captured. In high-parameter spectral panels, BV421, V450 and BV480 are often used together with careful marker assignments.