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Brilliant Violet Antibody Conjugates
  • Introduction to Brilliant Violet™ Antibodies

    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.

    Brilliant Violet™ fluorophore conjugated antibodies are a novel family of research reagents, co-developed with Sirigen, that will revolutionize flow cytometry. Excitable with the violet laser at 405 nm, these antibody reagents bring dramatic improvements in brightness and signal-to-noise over the current choices for the violet laser.
    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.

    Products
    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.

    Products
    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.

    Products
    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.

    Products
    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.

    Products
    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.

    Products
    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.

    Products

    Download the free Brilliant Violet Antibodies App to your iPhone or iPad today! Download the Brilliant Violet app today!
    Latest Publications

    • Tilton CA, et al. 2013. J Virol Methods. 166:376.  Pubmed
    • Chirkova T, et al. 2013. J. Virol. 87:13466.  Pubmed
    • Liu X, et al. 2013. Diabetes. 62:4228.  Pubmed
    • Bigley AB, et al. 2013. Brain Behav Immun. 889:1591.  Pubmed
    • Hardy CL, et al. 2013. J. Immunol. 191:5278.  Pubmed
    • Scally SW, et al. 2013. J. Exp Med. 210:2569.  Pubmed

    View more publications

    • Sugiyama D, et al. 2013. PNAS. 110:17945.  Pubmed
    • Vansaun MN, et al. 2013. PLoS One. 8:73054.  Pubmed
    • Toupadakis CA, et al. 2013. Cytotherapy. 15:1136.  Pubmed
    • van Aalderen MC, et al. 2013. J. Virol. 87:10263.  Pubmed
    • Caucheteux, SM, et al. 2013. PNAS. 10.1073/pnas.1221306110.  Pubmed
    • Chabtini, L, et al. 2013. J. Immunol. 190:88.  Pubmed
    • Takahashi, H, et al. 2012. Stem Cells Trans. Med. 1:803.  Pubmed
    • Canton, I, et al. 2013. FASEB J. 27:98.  Pubmed
    • Beres, AJ, et al. 2012. J. Immunol. 189:464.  Pubmed
    • Kinsey, GR, et al. 2012. J. Am. Soc. Nephrol. 10.1681/ASN.2012010070.  Pubmed
    • Xu, Y, et al. 2012. J. Immunol. 188:4113.  Pubmed
    • Chattopadhyay, PK, et al. 2012. Cytometry A. Apr 6. doi: 10.1002/cyto.a.22043.  Pubmed
    • Chattopadhyay, PK, and Roederer, M. 2012. Methods. doi: 10.1016/j.ymeth.2012.02.009.  Pubmed
    • Nijnik, A, et. al. Blood. 119:1370. Pubmed
    • Soghoian, DZ, et. al. Science Translational Medicine, 4:123ra25. Pubmed
    Brilliant Violet™, Brilliant Violet 421™, BV421™, Brilliant Violet 510™, BV510™, Brilliant Violet 570™, BV570™, Brilliant Violet 605™, BV605™, Brilliant Violet 650™, BV650™, Brilliant Violet 711™, BV711™, Brilliant Violet 785™, and BV785™ are trademarks of Sirigen Group Ltd.
    Watch the video tutorial on Brilliant Violet.
    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
    Learn the answers to Frequently Asked Questions about Brilliant Violet™ conjugates…
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  • 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.
    BV antibody conjugate figure

    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™ spectra
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  • Brilliant Flow Cytometry

    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.  For a guide on how to use Brilliant Violet™ conjugates in flow cytometry, download our technical sheet: Brilliant Violet™ Considerations for Multicolor Flow Cytometry.

    BV421 Stain Index 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.
    BV570 stain index 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.
    BV605 stain index 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.
    BV650 stain index 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.
    Brilliant Resolution
    CD127 data 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 data 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 data 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.
    Brilliant Viability: Nontoxic to Sorted Cells
    Nontoxic to 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.
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  • 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.

    Intravital Multiphoton Microscopy with BV421™ and BV510™
    Watch the Intravital Multiphoton Microscopy with BV421™ and BV510™ Video.
    Splenocytes were stained with CD4 BV421™ and CD8 BV510™ antibodies, and transferred back in vivo into mice. Mice were anesthetized, and had their lymph nodes surgically exposed. Exposed nodes were imaged using intravital multiphoton microscopy tuned to a single excitation wavelength of 830 nm according to established protocols. CD4 (blue) and CD8 (teal) T cells were imaged wandering in the node, exposures every 15 sec over a 30 minute period.

    Brilliant Stability
    Photostability curves 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.
    Photostability images
    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.
    CD56 Microscopy images 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 High Throughput
    BV421™ is useful for high thoughput content confocal imaging using the violet laser, providing clear and reliable results.
    High Throughput-Imaging 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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  • BV421™ BV570™ BV650™ BV785™ Mouse Human
    BV510™ BV605™ BV711™   Rat Isotype Control
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