Brilliant Violet™

Brilliant Violet™ antibody conjugates, proudly co-developed by BioLegend and Sirigen, are an innovative class of novel research reagents, providing more options for your multicolor flow cytometry panels and getting you better results. Maximize the capacity of your violet laser with our large selection of directly labeled Brilliant Violet™ antibody conjugates.

Features

• Extremely Bright
• Stable to Fixation
• Effective for Surface and Intracellular Antigens
• Exceptional Photostability
• Discrete Excitation/Emission Spectra
• No Special Disposal Requirements

• Nontoxic - for Sorting or Live Cell Imaging
• Effective Across a Wide pH Range
• No Special Buffers Required
• Large Selection of Antibody Specificities
• Excellent Pricing

Brilliant Violet™ Family

Brilliant Violet 421™

The first in the series, BV421™ can increase assay sensitivity by logarithmic orders of magnitude without increasing background or spillover, making it ideal for detecting rare cell populations or weakly expressed cell markers. It is exceptionally photostable, enabling the visualization of antigens with directly conjugated antibodies for confocal microscopy applications. Est. MW = 70 kD.

Excitation Max = 405 nm, Emission Max = 421 nm

Recommended filter = 450/50

Comparable Fluorophores: Pacific Blue™, Alexa Fluor® 405, eFluor® 450, BD Horizon™ V450, Cascade Blue™

Brightness = 5 (On a scale from 1 to 5, with 5 being the brightest.)

Molar Extinction Coeff. = 2,500,000 M-1cm-1

Quantum Yield = 0.65 in DPBS

Beta Testing -

Brilliant Violet 510™

Brilliant Violet 510™ is a novel non-tandem polymer with excellent signal-to-noise, excited by the violet laser. It can provide dramatic improvements over existing fluorophores emitting in this range such as Pacific Orange™, AmCyan, and Horizon™ V500. Est. MW = 77 kD.

Excitation Max = 405 nm, Emission Max = 510 nm

Recommended filter = 510/50

Comparable Fluorophores: BD Horizon™ V500, Pacific Orange™, Cascade Yellow™, AmCyan

Brightness = 2 (On a scale from 1 to 5, with 5 being the brightest.)

Molar Extinction Coeff. = 577,000 M-1cm-1

Quantum Yield = 0.44 in DPBS

Beta Testing -

Brilliant Violet 570™

Brilliant Violet 570™ is a novel molecule based on the Brilliant Violet 421™ polymer core. It provides a much brighter alternative to Pacific Orange™ for multicolor flow on the violet laser and is a better alternative to nanocrystals for intracellular flow cytometry. Est. MW = 60 kD.

Excitation Max = 405 nm, Emission Max = 570 nm

Recommended filter = 585/42

Comparable Fluorophores: Pacific Orange™, Cascade Yellow™, Qdot® 545, Qdot® 565, eFluor® 565NC

Brightness = 2 (On a scale from 1 to 5, with 5 being the brightest.)

Molar Extinction Coeff. = 2,300,000 M-1cm-1

Quantum Yield = 0.08 in DPBS

Beta Testing -

Brilliant Violet 605™

Brilliant Violet 605™ is a novel molecule based on the Brilliant Violet 421™ polymer core. It provides a much brighter alternative to eFluor® 605NC for multicolor flow on the violet laser and is a better alternative to nanocrystals for intracellular flow cytometry. Est. MW = 60 kD.

Excitation Max = 405 nm, Emission Max = 603 nm

Recommended filter = 610/20

Comparable Fluorophores: Qdot® 605, eFluor® 605NC

Brightness = 4 (On a scale from 1 to 5, with 5 being the brightest.)

Molar Extinction Coeff. = 2,400,000 M-1cm-1

Quantum Yield = 0.29 in DPBS

Beta Testing -

Brilliant Violet 650™

Brilliant Violet 650™ is a novel molecule based on the Brilliant Violet 421™ polymer core. It provides a much brighter alternative to eFluor® 650NC for multicolor flow on the violet laser and is a better alternative to nanocrystals for intracellular flow cytometry. Est. MW = 63 kD.

Excitation Max = 405 nm, Emission Max = 645 nm

Recommended filter = 660/20

Comparable Fluorophores: Qdot® 655, eFluor® 650NC

Brightness = 4 (On a scale from 1 to 5, with 5 being the brightest.)

Molar Extinction Coeff. = 2,500,000 M-1cm-1

Quantum Yield = 0.17 in DPBS

Beta Testing -

Brilliant Violet 711™

Brilliant Violet 711™ is a novel molecule based on the Brilliant Violet 421™ polymer core. It provides a much brighter alternative to eFluor® 700NC for multicolor flow on the violet laser and is a better alternative to nanocrystals for intracellular flow cytometry. Est. MW = 70 kD.

Excitation Max = 405 nm, Emission Max = 711 nm

Recommended filter = 710/50

Comparable Fluorophores: eFluor® 700NC, Qdot® 705

Brightness = 5 (On a scale from 1 to 5, with 5 being the brightest.)

Molar Extinction Coeff. = 2,800,000 M-1cm-1

Quantum Yield = 0.15 in DPBS

Beta Testing -

Brilliant Violet 750™

Brilliant Violet 750™ is a novel molecule based on the Brilliant Violet 421™ polymer core. It provides further options for the violet laser, particularly for those with either a spectral detection cytometer or a cytometer with a decagon configuration for the violet laser. Alternatively, it can be used in place of BV785™ on a standard violet laser octagon configuration. Est. MW = 70 kD.

Excitation Max = 405 nm, Emission Max = 750 nm

Recommended filter = 780/60 with a 740 LP

Comparable Fluorophores: None.

Brightness = 3 (On a scale from 1 to 5, with 5 being the brightest.)

Molar Extinction Coeff. = 2,301,131 M-1cm-1

Quantum Yield = 0.057 in DPBS

Brilliant Violet 785™

Brilliant Violet 785™ is a novel molecule based on the Brilliant Violet 421™ polymer core. It provides further options for the violet laser and is a better alternative to nanocrystals for intracellular flow cytometry. Est. MW = 60 kD.

Excitation Max = 405 nm, Emission Max = 785 nm

Recommended filter = 780/60

Comparable Fluorophores: QDot® 800

Brightness = 4 (On a scale from 1 to 5, with 5 being the brightest.)

Molar Extinction Coeff. = 2,500,000 M-1cm-1

Quantum Yield = 0.04 in DPBS

Beta Testing -

Cytokine profiling of T cell subsets using Brilliant Violet™ fluorophores

Learn the answers to Frequently Asked Questions about Brilliant Violet™ conjugates…

Brilliant Technology

The Brilliant Violet™ family of fluorescent molecules are organic polymers with an extraordinary capacity to absorb energy (extinction coefficient) and a high efficiency with which to convert that absorbed energy to an emitted signal (quantum yield). When conjugated to antibodies, this results in high intensity brightness on labeled cells.

Improvements over Traditional Organic Dyes

Traditional organic dyes are quite small, ranging between 300 - 1200 Da. Although multiple dyes can be conjugated to a single antibody, each acts independently from the others around them, thus their potency is limited to their own structural constraints and the potential for self quenching. In contrast, the Brilliant Violet™ polymers are comparable in size to AmCyan or APC, but unlike a protein, consist of repeating fluorescent subunits that act cooperatively along the entire length of the polymer backbone. Energy is conducted much like a molecular antennae to capture light and pass it down the antennae like a lightning rod. As the subunits work cooperatively, the polymers' extinction coefficient is substantially larger than that of standard organic dyes and this is reflected in superior brightness and signal-to-noise.

Brilliant Violet™ Chemistry

Structurally, Brilliant Violet™ polymers are unsaturated organic materials comprised of alternating single and double bonds and aromatic units. It is this repeating bond structure that creates a continuous π-orbital system and extended electronic delocalization. Such features give rise to unique and tunable optical properties, including large extinction coefficients (>10⁶ M^-1cm^-1), intense photoluminescence, and massive collective response, all of which help to address fundamental limitations in detection sensitivity. Adaptation of polymer side chain chemistry to impart solubility in aqueous solution confers ability for use as a highly sensitive fluorescent conjugate in biological applications such as flow cytometry and microscopy.

Physical properties, such as high quantum yield in typical flow buffers, high solubility, and minimal non-specfic binding, are all built into the backbone structure and in the side chain modifications. Additionally, the polymer design specifically incorporates well defined functional sites for covalent attachment to antibodies.

Brilliant Advantages

High Sensitivity Fluorescence™

Flourochromes derived from conducting polymers represent a new paradigm, High Sensitivity Fluorescence™, which aims to address fundamental shortcomings in reagents currently available for violet excitation in flow cytometry. Common dyes like Pacific Blue™, Alexa Fluor® 488 and Cy5 have high quantum yields but limited extinction coefficients. Alternative reporters like phycobiliproteins offer much greater absorbance cross-sections, producing brighter signals, but are limited by rapid photobleaching and sensitivity to fixation. Because the extinction coefficient of a conjugated polymer is directly proportional to the degree of polymerization (or number of repeat units), these structures are designed for improved brightness. Further, as these materials are derived from common synthetic organic and polymer chemistry techniques it is possible to manufacture reagents which are more defined and reproducible, in terms of size, conjugation sites, physical properties, and optical properties.
Unlike quantum dots, conjugated polymers have discrete excitation spectra, similar to that of organic dyes, which minimizes potential issues with cross-beam compensation.

Physical Properties

Brilliant Violet 421™ has an extinction coefficient of 2,500,000 M^-1cm^-1 at 405 nm, an aqueous solution quantum yield of 65 ± 5% and solubility in excess of 50 mg/mL in PBS. The extinction coefficient contributes to its superior brightness compared to Pacific Blue™, which has an extinction coefficient of 30,000 M^-1cm^-1. High Sensitivity Fluorescence™ polymers can also be modified with functional groups to produce high stokes shift emissions. Brilliant Violet 570™, Brilliant Violet 605™, and Brilliant Violet 650™ are such variants of the Brilliant Violet 421™ polymer, emitting maximally at 570 nm, 603 nm, and 645 nm, respectively. The figure below provides the excitation and emission spectra of the Brilliant Violet™ fluorophores. These can also be compared to other fluorophores in our Spectra Analyzer tool.

Brilliant Violet™antibody conjugates will change the future of flow cytometry, bringing new power to the violet laser. In particular, Brilliant Violet 421™ antibodies consistently stain at similar levels to PE, the brightest known fluorochrome, bringing unparalleled sensitivity and resolution to the violet laser, while Brilliant Violet 570™ antibodies add needed versatility to panel selection for multicolor flow cytometry. Brilliant Violet 605™ and Brilliant Violet 650™ antibodies also provide exceptionally bright signal and further expand options for multicolor panels. Brilliant Violet 711™ and Brilliant Violet 785™ bring newly expanded capability and selection for multicolor flow cytometry.

RBC-lysed human whole blood cells were stained with anti-CD3 conjugated to BV421™, PE, Pacific Blue™ or BD Horizon™ V450, and run on the BD™ LSR II flow cytometer. The stain index values indicated are derived at the optimal concentration for each conjugate. Stain index = (Median Fluorescence Intesity ^{positive cells} - Median Fluorescence Instensity ^{negative cells})/2 x rSD.

RBC-lysed human whole blood cells were stained with anti-CD8 conjugated to BV570™, Pacific Orange™ or BD Horizon™ V500, and run on the BD™ LSR II flow cytometer. The stain index values indicated are derived at the optimal concentration for each conjugate.

Human whole blood cells were stained with anti-CD8 conjugated to BV605™, or eFluor® 605NC, and run on the BD™ LSR II flow cytometer. The stain index values indicated are derived at the optimal concentration for each conjugate.

Human whole blood cells were stained with anti-CD8 conjugated to BV650™, or eFluor® 650NC, and run on the BD™ LSR II flow cytometer. The stain index values indicated are derived at the optimal concentration for each conjugate.

Easy to Use and Trouble-Free

Brilliant Violet™ antibodies are simple to use, compatible with standard staining buffers, and stable to fixation. Provided in convenient 5 µl test sizes at optimal ready-to-use concentrations, our Brilliant Violet™ antibody products can easily be added to your multi-color panels. To compare fluorescence spectra data of Brilliant Violet™ conjugates with our other fluorochromes use the BioLegend Fluorescence Spectra Analyzer.

Fluorescence Spectra Analyzer

To help with flow panel construction using BioLegend’s Brilliant Violet™ antibodies, use our Multicolor Panel Selector web tool.

Multicolor Panel Selector

Extensive Antibody Selection

BioLegend provides an expansive selection of antibody specificities for Brilliant Violet 421™, Brilliant Violet 570™, Brilliant Violet 605™, Brilliant Violet 650™, Brilliant Violet 711™, and Brilliant Violet 785™. All products are manufactured by our expert chemists in San Diego, CA and are supported by our 100% satisfaction guarantee. For samples or custom conjugations, contact our sales team.

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Brilliant Resolution

CD127

Human PBMCs were stained with anti-CD3 FITC and anti-CD127 (clone HCD127) conjugated to the BV421™ or Pacific Blue™. The data demonstrates the resolving power of BV421™, clearly showing separation of CD127-positive cells from dim and negative cells which can be difficult to define using Pacific Blue™

CD56

Human RBC-lysed whole blood cells were stained in an 11-color panel including HLA-DR PE/Cy7 and anti-CD56 (clone HCD56) conjugated to the BV421™ or anti-CD56 (clone MEM-188) Pacific Blue™. The data reveals discreet resolution of CD56 positive populations with BV421™. Data provided by Axel Schulz / Andreas Thiel, Charité - University Medicine Berlin.

Tetramer

Mouse spleen cells were labeled with PBS57-loaded mouse CD1d tetramer bound to Streptavidin-BV421™, -PE, or -Pacific Blue™. The data shows optimal staining with BV421™ when comparing equivalent tetramer concentration at 0.625 µg/ml. Data provided by Dr. Rick Willis, Emory/Yerkes. BioLegend does not currently offer catalog or custom CD1 tetramers.

Brilliant Viability: Nontoxic to Sorted Cells

Pooled Balb/c spleen and lymph node cells were stained with CD4-BV421™ or CD4-PB and sorted for CD4 positive cells. The cells were then labeled with CFSE and stimulated in 96-well flat-bottom plates coated with anti-CD3/anti-CD28 (1 µg/ml each) at 1 x 105 cells/well. On day 4, cells were analyzed for CFSE dilution. Figure A demonstrates comparable division and expansion of cells sorted by BV421™ compared to those sorted by PB, as indicated by the loss of CFSE signal. Unstimulated cells are displayed as the gray histogram. Figure B shows the median fluorescence values of each group. Error bars represent the standard deviation of triplicate wells. Figure C demonstrates that the % of cells that have divided is comparable between BV421™ and PB-stained cells. Overall, the data shows viability of cells after staining and sorting with a BV421™ antibody. Data provided by Aras Toker/Jochen Hühn, Helmholtz Centre for Infection Research.

Brilliant Options with Brilliant Violet 570™

Human RBC-lysed whole blood cells were stained with (A) anti-CD8 BV570™ and anti-CD56 BV421™ or (B) anti-CD127 BV570™ and anti-CD25 BV421™. In B, cells were gated on CD4 positive cells. The data demonstrates the capabilities for using BV570™ and BV421™ conjugates together in multicolor panels. Data in B, provided by Eva Tolosa, University Medical Center Hamburg-Eppendorf.

Brilliant Intracellular Detection with Brilliant Violet 570™

PMA/ionomycin-stimulated (6 hours) human peripheral blood lymphocytes were surface stained with CD3 FITC and then intracellularly stained with IFN-γ (clone 4S.B3) Brilliant Violet 570™ (right) or mouse IgG1, k Brilliant Violet 570™ isotype control (left).

Expanding Brilliance with Brilliant Violet 605™ and Brilliant Violet 650™

Human RBC-lysed whole blood cells were stained with anti-CD8 BV605™ and anti-CD3 BV650™. The data demonstrates distinct positive populations and low background negative populations.

Complete Brilliance with Brilliant Violet 711™ and Brilliant Violet 785™

RBC-lysed human whole blood cells were stained with CD127 BV785™ (clone A019D5) and CD25 BV711™ (clone BC96) to detect Treg cells.

Here at BioLegend, we are impartial when it comes to instrumentation for flow cytometry. Our reagents work beautifully on instruments from all manufacturers for diverse biological applications. But as this field of single cell, fluorescence-based multiplexing continues to grow in total multicolor capacity, sensitivity and ease of use, we can’t wait to release reagents to help match pace with these innovative instruments.

The Cytek Aurora™ Spectral Cytometer is one of these instruments. The introductory configuration of this instrument is a 3-laser system (405 nm, 488 nm and 633 nm) and provides the possibility for 48 different fluorescent parameters with 2 scatter channels. This is only the theoretical limit as we continue to strive to provide fluorophores that are distinct enough to be accurately unmixed on the system. The Aurora™ also utilizes APD (Avalanche Photodiode) detectors, which liberate the near-IR and infrared wavelengths for more efficient detection of emitted photons in this range.

We will expand on data generated in-house here at BioLegend in this section as we test the limits of spectral flow cytometry, but in the meantime, here is a glimpse of 21 color data from 3 lasers with the use of fluorophores that spectrally overlap almost identically on traditional machines. For example, in the panel to the right, PerCP and PerCP/Cy5.5, Alexa Fluor® 647 and APC, Brilliant Violet 421™ and Pacific Blue™ are all utilized at the same time. It also allows researchers to use eight Brilliant Violet™ (BV421™, BV510™, BV570™, BV605™, BV650™, BV711™, the new BV750™, and BV785™) fluorophores together with minimal compensation concerns. If you have additional questions on this panel, feel free to contact technical service.

(Right) Human whole blood was stained with 21 different antibody conjugates and analyzed on the Cytek Aurora™ Spectral Flow Cytometer.

Specificity	Format
CD141	APC
CD303	PE/Cy7
CD16	BV570™
CD14	PerCP/Cy5.5
CLEC9A	PE
CD1c	BV711™
IgD	BV510™
CD20	APC/Fire™ 750
CD4	APC-IR
CD8	Pacific Blue™
CD3	Alex Fluor® 700
CD25	BV421™
CCR6	BV785™
CD56	BV750™
CD45RA	FITC
CD27	PerCP
CD127	PE/Cy5
CD38	PE/Dazzle™ 594
HLA-DR	Alexa Fluor® 647
CD197	BV650™
CCR4	BV605™

T Cell Analysis

B Cell Analysis

Monocyte and Dendritic Cell Analysis

Brilliant Microscopy

Where BD Horizon™ V450, Pacific Blue™ and eFluor® 405 are just too dim for fluorescence microscopy, Brilliant Violet 421™ now enables visualization of antigens with directly conjugated antibodies, enhancing your capabilities with multi-color microscopy.

Historically, the "blue" channel on a microscope is reserved for an Hoechst or DAPI counterstaining due to the dim signal of typical dyes in that range, such as Alexa Fluor® 350, coumarin and Pacific Blue™ and the high sample autofluorescence often seen at that short wavelength of excitation. The following figures demonstrate that even on antibodies against markers that are relatively low in abundance, the Brilliant Violet 421™ direct conjugates can be used in imaging applications with high sensitivity. Further, they demonstrate extremely good photostability for repeated imaging and confocal z-stack capture. Also, these direct conjugates of Brilliant Violet 421™ demonstrate that there is no need for secondary antibodies or other amplificatiton techniques in order to achieve sufficient signal. Brilliant Violet 421™ will enable the addition of another color to a multicolor microscopy application on instruments capable of exciting this fluorophore between 360-420 nm.

Brilliant Stability

Figure A. Photostability Curves plotting Brilliant Violet 421™ with and without antifade in the mounting medium against Pacific Blue™ mounted with no antifade. Photobleaching was conducted on a 3i spinning disk confocal, laser power at 100%, 300ms exposures every 1 s for a duration of 130 seconds. The curve is plotted as percent fluorescence relative to the initial fluorescence intensity. Since data is normalized to 100%, it is not a reflection on the initial fluorescence intensity for each fluorophore as BV421™ is much brighter than Pacific Blue™, as demonstrated in Figure B. With or without antifade, BV421™ retains more than half-maximal signal even after 130 seconds. Pacific Blue™, on the other hand, loses fluorescence more quickly, showing only 50% intensity by 60 seconds. Prolong Gold was able to attenuate the loss of fluorescence intensity for BV421™ upon initial exposure to laser, particularly noticeable within the first 10 to 30 seconds. Since most images are typically acquired within that initial few seconds, it is recommended to use a mounting medium containing antifade to preserve the brightest signal.

Figure B. Still images were captured at 0, 10, 20, 60, and 120 seconds. Samples were human PBMCs labeled with Brilliant Violet 421™ CD3 or Pacific Blue™ CD45. CD45 was required for detection of Pacific Blue™ because of its higher abundance than CD3 on PBMCs. Even though Pacific Blue™ endured 60 seconds of exposure before it lost 50% of its intensity, it was no longer bright enough at 20 seconds to be useful in imaging. The Brilliant Violet™ conjugates, on the other hand, were extremely bright and stable with antifade. Even at 120 seconds, a useful image could still be captured when antifade was used.

Brilliant Imaging

For multi-color imaging, the use of directly conjugated antibodies is essential. Brilliant Violet™ brings new capabilities to the violet laser, allowing for bright staining of weakly expressed markers.

NK92 cells (human NK cell line) were fixed and stained with anti-CD56 BV421, anti-perforin FITC, and phalloidin AF568 imaged on an Olympus IX81 spinning disk confocal microscope on 100X objective, NA 1.45. Exposures: 488 = 1000 ms, 568 = 100 ms, BV421™ (450 nm) = 200 ms. Data provided by Emily Mace and Jordan Orange, University of Pennsylvania.

Brilliant Violet 421™ Filter Choice

As previously mentioned, BV421™ is used in the “blue” channel, which is typically occupied by DAPI or Alexa Fluor® 405. However, as there is no set BV421™ filter on the market yet and because a “DAPI filter” may not be ideal, filter choice based on their wavelengths (excitation, emission and dichroic filters) is particularly important.

For example, the filter set in the image below works well as the dichroic filter (red line) does not block any of the BV421™ emission. However, in filter setups where this dichroic is set further to the right, a large percentage of the photons emitted by BV421™ may not reach the camera. If your filter setup is suitable, BV421™ can provide you with a bright, photostable option for multicolor microscopy.

BioLegend has validated the following filter setups for BV421™ (Ex: Excitation, Em: Emission):

Ex: 379/34, Dichroic: 409 LP, Em: 425/26
Ex: 395/25, Dichroic: 405 LP, Em: 440/40
Ex: 350/50, Dichroic: 400 LP, Em: 460/50

Brilliant High Throughput

BV421™ is useful for high thoughput content confocal imaging using the violet laser, providing clear and reliable results.

Primary human macrophages in 384-well plates were polarized to M1 or M2, then fixed and stained with Brilliant Violet 421™ CD38. Plate was read on the ImageXpress Ultra from Molecular Devices, and fluorescence signal was quantitated across 6 replicates. The mean fluorescence + SD is plotted. Data provided by unnamed collaborator.

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