Multicolor Flow Cytometry

Video Library

New Releases

Design

Quality flow cytometry data starts with proper panel design. Fluorochrome choices, instrument configurations/setup and knowledge of the biology being studied are important factors when considering panel choices.

In this module, we will provide you with a foundation to easily build the best possible panels to answer your experimental questions.

Analyze

A good panel is defined by its ability to resolve populations of interest. Matching bright fluorochromes to dim markers, and the ability to spread markers across multiple lasers, can minimize spectral overlap and maximize population resolution.

In the Analyze section, multiple data sets demonstrating how to maximize resolution of cell populations will be shared. Also included is an interactive session to review panels that you helped design.

Sort

The ability to sort highly pure cell populations is fundamental to downstream biological analysis. An optimized panel is a key component of sort purity.

The Sort module reviews important considerations when you move from cell analysis to cell sorting. Information on how optimized panels improve sort outcomes will be discussed.

All Horizon Videos

Reveal

The journey to “see what nature is hiding” using flow cytometry begins by understanding the biology of your cells and the relative brightness of fluorochromes. To maximize resolution, bright fluorochromes are used to resolve dim antigens. Enabling brighter fluorescence signals, BD Horizon Brilliant™ polymer dyes are perhaps the most significant advancement in reagent technology in a decade.

Elevate

By understanding antigen density—the number of cell surface receptors on a cell—researchers can understand which antibody/fluorochrome combinations are candidates for panels.

Resolve

Delivering information from multiple parameters simultaneously, a single flow cytometry experiment can speed the path to discovery. Traditionally, this is accomplished using many fluorochromes on a few lasers and subsequently addressing spectral overlap using compensation. The more colors used, the more overlap and the more complex the experiment.

Achieve

Biology is seldom simple, but by leveraging new tools and methods, you can speed your journey to see nature more clearly. Our single goal at BD Biosciences is to equip you with the ability to be more efficient and effective in your research by making flow cytometry more powerful and easier to use.

Sentinel

Discover a new way of looking at fluorochromes and markers that can maximize the number of parameters achievable on any instrument.

Explore the Latest in Flow Cytometry Technology

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:

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:

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:

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:

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:

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:

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:

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:

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:

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:

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:

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:

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:

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:

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)

PE-Cy™5

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:

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:

APC-Cy7: US Patent 5,714,386

Featured Product

Flow Cytometry Analyzers

Application Notes

Brochures

• BD Horizon Brilliant™ Dyes

Catalogs

• BD Multicolor Antibody Reagents Catalog

Experiment Sheets

• BD LSRFortessa™ X-20 15-Color Immunophenotyping
• BD LSRFortessa™ X-20 System 5-Color Panels Designed for Minimal Compensation

Product Lists

• Mouse Research Reagents Product List
• NA/LE Reagents Product List
• Small Size Flow Cytometry Reagents Product List