Mulitcolor Flow Cytometry

Empower your multicolor panel design with BD tools and information assembled here to help you take full advantage of the power of multicolor flow cytometry. Our comprehensive portfolio of conjugated antibodies is designed to help characterize cells through surface, intracellular, or secreted markers.

The portfolio includes BD Horizon Brilliant TM dyes that enable resolution of cell populations previously obscured, opening new avenues of investigation using flow cytometry.

BD Horizon Brilliant Dyes

Ultraviolet Laser Violet Laser Blue Laser Green/Yellow-Green Laser Red Laser

Fluorochromes

BD Horizon™ BUV395

BD Horizon Brilliant™ Ultraviolet 395 (BUV395) (Ex Max 348 nm/Em Max 395 nm) is a UV-excitable dye that has been developed exclusively by BD Biosciences for instruments equipped with a 355-nm UV laser. This dye is optimal for multicolor flow cytometry because it has little to no spillover into other detectors.

Filter: 379/28
Relative brightness: Moderate (on a scale of Dim, Moderate, Bright, and Brightest)
Sample data:

Lysed whole blood stained with CD4 or CD56 in BUV395 or FITC formats

BD Horizon™ BUV496

BD Horizon Brilliant™ Ultraviolet 496 (BUV496) (Ex Max 348 nm/Em Max 496 nm) is a tandem fluorochrome that combines BD Horizon BUV395 and an acceptor dye with an emission maximum at 496 nm. Due to the excitation of the acceptor dye by other laser lines, there may be significant spillover into the channel detecting BD Horizon™ V500 or BV510 (for example, 525/40-nm filter). BUV496 has been exclusively developed by BD Biosciences for instruments equipped with a 355-nm UV laser.

Filter: 515/30
Relative brightness: Moderate (on a scale of Dim, Moderate, Bright, and Brightest)
Sample data:

Lysed whole blood stained with CD4 or CD19 in BUV496 or FITC formats.

BD Horizon™ BUV563

BD Horizon™ BUV563 is a tandem fluorochrome that combines BD Horizon BUV395 and an acceptor dye with an Em Max at 563 nm. Due to the excitation of the acceptor dye by other laser lines, there may be spillover into channels detecting PE (eg, 575/26-nm filter) and PE-CF594 (eg, 610/20-nm filter). BUV563 has been exclusively developed by BD Biosciences for instruments equipped with a 355-nm UV laser.

Filter: 585/15
Relative brightness: Bright (on a scale of Dim, Moderate, Bright, and Brightest)
Sample data:

Flow cytometric analysis of CD19 expression on human peripheral blood lymphocytes.

Two-color flow cytometric analysis of CD4 expression on mouse splenocytes.

BD Horizon™ BUV661

BD Horizon Brilliant™ Ultraviolet 661 (BUV661) (Ex Max 348 nm/Em Max 661 nm) is a tandem dye that combines BUV395 and an acceptor dye with an emission maximum at 661 nm. As part of the BD Horizon Brilliant Ultraviolet family, BUV661 provides an additional option for multicolor panels utilizing UV excitable dyes.

Filter: 670/25, 630nm
Relative brightness: Bright (on a scale of Dim, Moderate, Bright, and Brightest)
Sample data:

Lysed whole blood stained with Hu CD3 or HU CD11c BUV661 and appropriate isotype control.

Mouse splenocytes stained with Ms CD3e Alexa Fluor® 488 and Ms CD19 BUV661

Mouse bone-marrow cells stained with Ms CD11b BUV661.

BD Horizon™ BUV737

BD Horizon Brilliant™ Ultraviolet 737 (BUV737) (Ex Max 348 nm/Em Max 737 nm) is a tandem fluorochrome that combines BD Horizon BUV395 and an acceptor dye with an emission maximum at 737 nm. Due to the excitation of the acceptor dye by other laser lines, there may be significant spillover into channels detecting dyes with properties like Alexa Fluor® 700 (for example, 712/20-nm filter). BUV737 has been exclusively developed by BD Biosciences for instruments equipped with a 355-nm UV laser.

Filter: 740/35
Relative brightness: Bright (on a scale of Dim, Moderate, Bright, and Brightest )
Sample data:

Lysed whole blood stained with CD19 or CD127 in BUV737 or FITC formats

BD Horizon™ BUV805

BD Horizon Brilliant™ Ultraviolet 805 (BUV805) (Ex Max 348 nm/Em Max 805 nm) is a tandem fluorochrome that combines BD Horizon BUV395 and an acceptor dye with an emission maximum at 805 nm. BUV805 has been exclusively developed by BD Biosciences for instruments equipped with a 355-nm UV laser.

Filter: 820/60
Relative brightness: Dim (on a scale of Dim, Moderate, Bright, and Brightest)
Sample data:

Lysed whole blood stained with CD4 or CD20 BUV805 and appropriate isotype control.

Fluorochromes

BD Horizon™ BV421

BD Horizon Brilliant™ Violet 421 (BV421) (Ex-Max 407 nm/Em-Max 421 nm) is a polymer-based dye excited by the violet laser and is one of the brightest fluorochromes offered by BD Biosciences. Conjugates are typically 10 times brighter than Pacific Blue™ conjugates and are often as bright as or brighter than PE conjugates. Due to nearly identical excitation and emission properties but different spillover characteristics, BV421, Pacific Blue™, and BD Horizon™ V450 cannot be used simultaneously.

Filter: 450/40
Relative brightness: Brightest (on a scale from Dim, Moderate, Bright, and Brightest)
Sample data:

Comparison data from two regulatory T-cell panels

T-cell subset resolved: CD8 memory and effector T cells

T-cell subset resolved: CD4 memory and effector T cells

T-cell subset resolved: Regulatory T cells

Example data from a 7-color TNK panel

Example of BV421 used in an intracellular staining protocol

Example of BV421 staining using various protocols

Analysis of human T-cell subsets

Various subsets of regulatory T cells

Memory/effector CD4+ and CD8+ T cells

BD Horizon™ BV510

BD Horizon Brilliant™ Violet 510 (BV510) (Ex-Max 405 nm/Em-Max 510 nm) is a polymer-based dye. BD Horizon BV510 can be excited by the violet laser and detected in the BD Horizon V500 (525/50-nm) filter set. Due to similar excitation and emission properties, BD Horizon BV510 and BD Horizon V500 cannot be used simultaneously.

Filter: 525/50
Relative brightness: Moderate (on a scale from Dim, Moderate, Bright, and Brightest)
Sample data:

CD4 BD Horizon V500 comparison to Pacific Orange™

Analysis of human lymphocytes: T-cell subsets

Analysis of human lymphocytes: Non–T-cell subsets

BD Horizon™ BV510

BD Horizon Brilliant™ Violet 510 (BV510) (Ex-Max 405 nm/Em-Max 510 nm) is a polymer-based dye. BD Horizon BV510 can be excited by the violet laser and detected in the BD Horizon V500 (525/50-nm) filter set. Due to similar excitation and emission properties, BD Horizon BV510 and BD Horizon V500 cannot be used simultaneously.

Filter: 525/50
Relative brightness: Moderate (on a scale from Dim, Moderate, Bright, and Brightest)
Sample data:

Example data for CD11c BV510 and CD19 BV510

Stain index comparison between BD Horizon BV510 and FITC

BD Horizon™ BV605

BD Horizon Brilliant™ Violet 605 (BV605) (Ex-Max 407 nm/Em-Max 602 nm) is a tandem fluorochrome that combines BD Horizon BV421 and an acceptor dye. These conjugates are very bright, exhibiting brightness similar to equivalent PE conjugates. Due to the excitation of the acceptor dye by the green (532-nm) and yellow-green (561-nm) lasers, there will be significant spillover of the BV605 signal into the PE and BD Horizon™ PE-CF594 detectors off the green or yellow-green lasers.

Filter: 610/20
Relative brightness: Bright (on a scale from Dim, Moderate, Bright, and Brightest)
Sample data:

Example data for BV605

Example data from a human hematopoietic stem cell panel

Example of BV605 staining using various protocols

BD Horizon™ BV650

BD Horizon Brilliant™ Violet 650 (BV650) (Ex-max 407 nm/Em-max 650 nm) is a tandem fluorochrome of BD Horizon BV421 and an acceptor dye with an Em Max at 650 nm. Due to the excitation and emission characteristics of the acceptor dye, there will be spillover into the APC and Alexa Fluor® 700 detectors. BD Horizon BV650 will have moderate spillover into the BD Horizon™ BV711 detector.

Filter: 660/20
Relative brightness: Brightest (on a scale from Dim, Moderate, Bright, and Brightest)

 

 

BD Horizon™ BV711

BD Horizon Brilliant™ Violet 711 (BV711) (Ex-Max 407 nm/Em-Max 711 nm) is a tandem fluorochrome of BD Horizon BV421 and an acceptor dye with an Em Max at 711 nm. This dye offers a very bright choice for the violet laser. Due to the excitation and emission characteristics of the acceptor dye, there may be moderate spillover into the Alexa Fluor® 700 and PerCP-Cy™5.5 detectors.

Filter: 710/50
Relative brightness: Brightest (on a scale from Dim, Moderate, Bright, and Brightest)
Sample data:

Example data for CD335 (NKp46) BV711 and CD19 BV711

Various subsets of memory/effector CD4+ and CD8+ T cells

BD Horizon Brilliant Violet dyes detecting various subsets of CD8+ T cells

BD Horizon Brilliant Violet dyes detecting various subsets of memory/effector CD4+ T cell

BD Horizon™ BV786

BD Horizon Brilliant™ Violet 786 (BV786) (Ex-max 407 nm/Em-max 786 nm) is a tandem fluorochrome of BD Horizon BV421 and an acceptor dye with an Em Max at 786 nm. BD Horizon™ BV786 offers a bright choice for the 6th detector off the violet laser.

Filter: 780/60
Relative brightness: Bright (on a scale from Dim, Moderate, Bright, and Brightest)

Fluorochromes

BD Horizon™ BB515

BD Horizon Brilliant™ Blue 515 (BB515) (Ex-Max 490 nm/Em-Max 515 nm) is a dye that was exclusively developed by BD Biosciences as a brighter alternative to FITC. This dye is up to seven times brighter than FITC and has less spillover into the PE channel. Due to similar excitation and emission properties, BB515, FITC, and Alexa Fluor® 488 cannot be used simultaneously.

Filter: 530/30
Relative brightness: Brightest (on a scale from Dim, Moderate, Bright, and Brightest)
Sample data:

Lysed whole blood stained with Hu CD3 or CD19 FITC (red), BB515 (blue), or Alexa Fluor® 488 (green)

Whole blood stained with Hu CD4 APC, CD127 PE, CD3 PerCP-Cy™5.5, and CD25 FITC or CD25 BB515

BD Horizon™ BB700

BD Horizon Brilliant™ Blue 700 (BB700) (Ex-Max 488 nm/Em-Max 693 nm) is a dye that was exclusively developed by BD Biosciences as a brighter alternative to PerCP-Cy5.5, making it better suited for resolving dim populations. This dye also has less cross laser excitation off the 405 nm laser, resulting in less spillover into the violet channels compared to PerCP-Cy5.5. Due to similar excitation and emission properties, BD Horizon BB700 and PerCP-Cy5.5 cannot be used simultaneously.

Filter: 695/40
Relative brightness: Brightest (on a scale from Dim, Moderate, Bright, and Brightest)
Sample data:

BD Horizon Brilliant™ Blue 700 Human CD4 Comparison

FITC

FITC (Ex-Max 494 nm/Em-Max 520 nm), fluorescein isothiocyanate, is a fluorochrome with a molecular weight of 389 Da. FITC is sensitive to pH changes and photobleaching. Due to nearly identical excitation and emission properties but different spillover characteristics, FITC and Alexa Fluor® 488 cannot be used simultaneously. FITC is relatively dim and should be reserved for highly expressed markers whenever possible.

Filter: 530/30
Relative brightness: Moderate (on a scale from Dim, Moderate, Bright, and Brightest)

 

Alexa Fluor® 488

Alexa Fluor 488 (Ex-Max 495 nm/Em-Max 519 nm) conjugates are highly photostable and remain fluorescent over a broad pH range. The excitation and emission maxima are nearly identical to those of FITC. However, Alexa Fluor 488 tends to be brighter and more optimal for intracellular applications. Due to nearly identical excitation and emission properties but different spillover characteristics, FITC and Alexa Fluor 488 cannot be used simultaneously. Alexa Fluor 488 exhibits extraordinary photostability, which makes it highly suitable for fluorescence microscopy.

Filter: 530/30
Relative brightness: Moderate (on a scale from Dim, Moderate, Bright, and Brightest)

 

PE

PE (Ex-Max 496 nm/Em-Max 578 nm), R-phycoerythrin (PE), is an accessory photosynthetic pigment found in red algae. It exists in vitro as a 240-kDa protein with 23 phycoerythrobilin chromophores per molecule. This makes PE the brightest fluorochrome for flow cytometry applications, but its photobleaching properties make it unsuitable for fluorescence microscopy.

Filter: 575/26
Relative brightness: Bright (on a scale from Dim, Moderate, Bright, and Brightest)

 

BD Horizon™ PE-CF594

BD Horizon PE-CF594 (Ex-Max 496 nm/Em-Max 612 nm) is a tandem conjugate, developed exclusively by BD Biosciences, that combines PE and CF594. PE-CF594 is a brighter alternative to PE-Texas Red®, with improved spectral characteristics. PE-CF594 reagents exhibit very consistent spillover values lot-to-lot, making them an ideal choice for the PE-Texas Red® detector (610/20 nm).

Filter: 610/20
Relative brightness: Brightest (on a scale from Dim, Moderate, Bright, and Brightest)
Sample data:

Staining comparison of BD Horizon PE-CF594, ECD, and PE-Alexa Fluor® 610

Example of BD Horizon PE-CF594 staining using various protocols

Example data from a 10-color TBMNK panel

Example data from a 9-color memory/effector T-cell panel

PE-Cy™5

PE-Cy5 (Ex-Max 496 nm/Em-Max 667 nm) is a tandem conjugate that combines phycoerythrin and a cyanine dye. Because of its broad absorption range and the fact that its emission spectra are equivalent to APC, PE-Cy5 is not recommended for simultaneous use with APC. The Cy5 molecule has been shown to exhibit nonspecific binding to Fc receptors, which is most apparent on monocyte populations. PE-Cy5 is not recommended for fluorescence microscopy because it is subject to photobleaching.

Filter: 670/14
Relative brightness: Brightest (on a scale from Dim, Moderate, Bright, and Brightest)

 

PerCP

PerCP (Ex-Max 482 nm/Em-Max 678 nm) is a component of the photosynthetic apparatus found in the dinoflagellate Glenodinium. PerCP is a protein complex with a molecular weight of approximately 35 kDa. Due to its photobleaching characteristics, PerCP conjugates are not recommended for use on flow cytometers with high-power lasers (>25 mW).

Filter: 695/40
Relative brightness: Dim (on a scale from Dim, Moderate, Bright, and Brightest)

 

PerCP-Cy™5.5

PerCP-Cy5.5 (Ex-Max 482 nm/Em-Max 695 nm) is a tandem conjugate that combines PerCP with a cyanine dye. PerCP-Cy5.5 is not subject to photobeaching like PerCP and can be used with stream-in-air flow cytometers. It has less Fc receptor-mediated nonspecific staining than PE-Cy5. Additionally, the PerCP-Cy5.5 tandem conjugate is not as susceptible to fixative or light instability as APC-Cy7 and PE-Cy7.

Filter: 695/40
Relative brightness: Moderate (on a scale from Dim, Moderate, Bright, and Brightest)

 

PE-Cy™7

PE-Cy7 (Ex-Max 496 nm/Em-Max 785 nm) is a tandem fluorochrome that combines PE and a cyanine dye. PE-Cy7 conjugated reagents are as bright as PE conjugates. PE-Cy7 is particularly sensitive to photo-induced degradation, resulting in loss of fluorescence and changes in fluorescence spillover. Extreme caution must be taken to avoid light exposure and prolonged exposure to paraformaldehyde fixative. Fixed cells should be analyzed within 4 hours of fixation in paraformaldehyde or transferred to a paraformaldehyde-free buffer for overnight storage.

Filter: 780/60
Relative brightness: Bright (on a scale from Dim, Moderate, Bright, and Brightest)

Fluorochromes

PE

PE (Ex-Max 496 nm/Em-Max 578 nm), R-phycoerythrin (PE), is an accessory photosynthetic pigment found in red algae. It exists in vitro as a 240-kDa protein with 23 phycoerythrobilin chromophores per molecule. This makes PE the brightest fluorochrome for flow cytometry applications, but its photobleaching property make it unsuitable for fluorescence microscopy.

Filter: 575/26
Relative brightness: Brightest (on a scale from Dim, Moderate, Bright, and Brightest)

 

BD Horizon™ PE-CF594

BD Horizon PE-CF594 (Ex-Max 496 nm/Em-Max 612 nm) is a tandem conjugate, developed exclusively by BD Biosciences, that combines PE and CF594. PE-CF594 is a brighter alternative to PE-Texas Red®, with improved spectral characteristics. PE-CF594 reagents exhibit very consistent spillover values lot-to-lot, making them an ideal choice for the PE-Texas Red® detector (610/20 nm).

Filter: 610/20
Relative brightness: Brightest (on a scale from Dim, Moderate, Bright, and Brightest)

Staining comparison of BD Horizon PE-CF594, ECD, and PE-Alexa Fluor® 610

Example of BD Horizon PE-CF594 staining using various protocols

Example data from a 10-color TBMNK panel

Example data from a 9-color memory/effector T-cell panel

PE-Cy™5

PE-Cy5 (Ex-Max 496 nm/Em-Max 667 nm) is a tandem conjugate that combines phycoerythrin and a cyanine dye. Because of its broad absorption range and the fact that its emission spectrum is equivalent to APC, PE-Cy5 is not recommended for simultaneous use with APC. The Cy5 molecule has been shown to exhibit nonspecific binding to Fc receptors, which is most apparent on monocyte populations. PE-Cy5 is not recommended for fluorescence microscopy because it is subject to photobleaching.

Filter: 670/14
Relative brightness: Brightest (on a scale from Dim, Moderate, Bright, and Brightest)

 

PE-Cy™7

PE-Cy7 (Ex-Max 496 nm/Em-Max 785 nm) is a tandem fluorochrome that combines PE and a cyanine dye. PE-Cy7 conjugated reagents are as bright as PE conjugates. PE-Cy7 is particularly sensitive to photo-induced degradation, resulting in loss of fluorescence and changes in fluorescence spillover. Extreme caution must be taken to avoid light exposure and prolonged exposure to paraformaldehyde fixative. Fixed cells should be analyzed within 4 hours of fixation in paraformaldehyde or transferred to a paraformaldehyde-free buffer for overnight storage.

Filter: 780/60
Relative brightness: Brightest (on a scale from Dim, Moderate, Bright, and Brightest)

Fluorochromes

APC

Allophycocyanin (APC) (Ex Max 650 nm/Em Max 660 nm) is an accessory photosynthetic pigment found in blue-green algae. Its molecular weight is approximately 105 kDa. APC has six phycocyanobilin chromophores per molecule, which make it a very bright fluorochrome that is highly suitable for flow cytometry applications. Due to nearly identical excitation and emission properties but different spillover characteristics, APC and Alexa Fluor® 647 cannot be used simultaneously.

Filter: 660/20
Relative brightness: Bright (on a scale of Dim, Moderate, Bright, and Brightest)

 

Alexa Fluor® 647

Alexa Fluor® 647 (Ex Max 650 nm/Em Max 668 nm) conjugates are highly photostable and remain fluorescent over a broad pH range. The excitation and emission maxima are nearly identical to those of APC. However, APC tends to be brighter while Alexa Fluor® 647 is more optimal for intracellular applications. This fluorochrome exhibits uncommon photostability, making it an ideal choice for use in fluorescence microscopy. Due to nearly identical excitation and emission properties but different spillover characteristics, APC and Alexa Fluor® 647 cannot be used simultaneously.

Filter: 660/20
Relative brightness: Bright (on a scale of Dim, Moderate, Bright, and Brightest)

BD Horizon™ APC-R700

BD Horizon™ APC-R700 (Ex Max 652 nm/Em Max 704 nm) is a tandem fluorochrome that combines APC with R700, a proprietary organic dye. This dye has been developed exclusively by BD Biosciences as a brighter alternative to Alexa Fluor® 700. Due to similar excitation and emission properties, APC-R700 and Alexa Fluor® 700 cannot be used simultaneously.

Filter: 730/45
Relative brightness: Bright (on a scale of Dim, Moderate, Bright, and Brightest)
Sample Data:

Cells stained with APC-R700 show a large spread between positive and negative populations similar to cells stained with PE

Alexa Fluor® 700

Alexa Fluor® 700 (Ex Max 696 nm/Em Max 719 nm) is a far-red dye that can be excited with a 633–640-nm laser. This enables multicolor analysis in conjunction with APC and Alexa Fluor® 647, or APC-H7 and APC-Cy™7 reagents.

Filter: 730/45
Relative brightness: Dim (on a scale of Dim, Moderate, Bright, and Brightest)

 

APC-Cy™7

APC-Cy7 (Ex Max 650 nm/Em Max 785 nm) is a tandem fluorochrome that combines APC and a cyanine dye (Cy7). Special precautions must be taken with APC-Cy7 conjugates, and cells stained with them, to protect the fluorochrome from long-term exposure to light. Some APC-Cy7 conjugates show changes in their emission spectra with prolonged exposure to paraformaldehyde. Fixed cells should be analyzed within 4 hours of fixation in paraformaldehyde or transferred to a paraformaldehyde-free buffer for overnight storage. Due to nearly identical excitation and emission properties but different spillover characteristics, APC-Cy7 and APC-H7 cannot be used simultaneously.

Filter: 780/60
Relative brightness: Dim (on a scale of Dim, Moderate, Bright, and Brightest)

 

BD™ APC-H7

BD APC-H7 (Ex Max 650 nm/Em Max 785 nm) is an APC-cyanine tandem fluorochrome, which uses an analog of Cy7 and has similar spectral properties to APC-Cy7. APC-H7 conjugates provide greater stability in light and paraformaldehyde fixatives and have less spillover into the APC channel than APC-Cy7 conjugates. APC-H7 conjugates are typically 75% as bright as equivalent APC-Cy7 conjugates. Due to nearly identical excitation and emission properties but different spillover characteristics, APC-Cy7 and APC-H7 cannot be used simultaneously.

Filter: 780/60
Relative brightness: Dim (on a scale of Dim, Moderate, Bright, and Brightest)
Sample data:

Red Laser - BD APC-H7

APC-Cy7: US Patent 5,714,386


For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Pacific Blue™ and Pacific Orange™ are trademarks, and Alexa Fluor® and Texas Red® are registered trademarks of Life Technologies Corporation.

Cy™ is a trademark of GE Healthcare. Cy™ dyes are subject to proprietary rights of GE Healthcare and Carnegie Mellon University and are made and sold under license from GE Healthcare only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from GE Healthcare, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.

CF™ is a trademark of Biotium, Inc.

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