Flow Cytometry

Antibodies Conjugated to Fluorophores

Bio-Rad offers a wide range of antibodies conjugated to the most commonly used fluorophores. In addition to FITC, PE and APC, we also offer Alexa Fluor and DyLight conjugates in an array of colors, both with outstanding spectral properties, suitable for use in flow cytometry, fluorescence microscopy, and fluorescent western blotting.

If you can’t find the labeled version you need, our easy-to- LYNX Rapid Conjugation Kits®  for many popular dyes and tandem conjugates, and Readilink Antibody Conjugation Kits for flow cytometry optimized fluorophores can help.

Check out our new StarBright Fluorophores developed for flow cytometry

Antibodies Conjugated to Fluorochromes

Antibodies Conjugated to Fluorophores

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Fluorophores for Flow Cytometry

Not all fluorophores are suitable for all antibody based applications. For example, even though PE is bright, it is easily bleached and is therefore not suitable for fluorescence imaging. Due to the increase in the size of flow cytometry panels, flow cytometry requires bright fluorophores that have specific excitation and emission wavelengths from one laser. The fluorophores must be compatible with each other in the panel, and ideally should be photostable, with no loss of fluorescence when fixed.

Visit bio-rad-antibodies.com/flow-cytometry-fluorophores for more detailed information on flow cytometry compatible fluorophores, including our new proprietary StarBright Fluorophores

Single Dyes 

Fluorophore Fluorescence color Maximum excitation (nm) Maximum emission (nm)
Spectrally similar dyes
DyLight 405   400 420 3 Alexa Fluor 405, Cascade Blue
Alexa Fluor 405   401 421 3  
Pacific Blue   410 455 1  
DyLight 488   493 518 4 Alexa Fluor 488, FITC
Alexa Fluor 488   495 519 3 Cy2, DyLight 488, FITC
FITC   490 525 3 Alexa Fluor 488, Cy2, DyLight 488
DyLight 550   562 576 4 Alexa Fluor 546, Alexa Fluor 555, Cy3, TRITC
PE   496, 546 578 5  
hFab™ Rhodamine*   530 580 3  
Texas Red   596 615 2  
APC   650 661 4 Alexa Fluor 647, Cy5
Alexa Fluor 647   650 665 4 APC, Cy5, DyLight 650
Cy5   649 670 3  
DyLight 650   654 673 4 Alexa Fluor 647, Cy5
PerCP   490 675 2  
StarBright™ Blue 700*   470 700 5  
DyLight 680   692 712 4 Alexa Fluor 680, Cy5.5
Alexa Fluor 700 Infrared 702 723 2  
DyLight 755 Infrared 752 778 4 Alexa Fluor 750
DyLight 800* Infrared 777 794 4 IR Dye 800

* Western blot tested

Abbreviations: APC; allophycocyanin, FITC; fluorescein isothiocyanate, PE; phycoerythrin (Note. phycoerythrin (PE) is the same as R-phycoerythrin (RPE)), PerCP; peridinin-chlorophyll-protein complex.

Tandem Dyes for Flow Cytometry

When designing panels of eight or more colors, tandem dyes, such as APC-Cy7, have to be included. This is due to both laser excitation and single fluorophores limitations, which make it necessary for a single laser to excite the maximum number of fluorophores possible. 

Tandem dyes, as the name implies, consist of two different covalently attached fluorophores (a donor and an acceptor molecule). With regards to spectral properties, the tandem dye has the excitation characteristics of the donor fluorophores and the emission characteristics of the acceptor molecule. These properties are due to Förster resonance energy transfer (FRET; also known as Fluorescence resonance energy transfer); a process in which energy is passed on from an excited donor to a nearby acceptor molecule, which then emits a photon of light.

For tandem dyes the following guidelines should be followed:

  • Tandem dyes are highly susceptible to photobleaching and therefore need to be protected from light at all times.
  • Tandem dye antibody conjugates should never be frozen. Freezing might result in denaturing of the donor fluorophores and thereby no or reduced staining.
  • The brightness of tandem dyes might be reduced by a fixation or permeabilization step. If fixation or permeabilization is required, the duration should be as short as possible. 
  • Tandem dyes have a high batch-to-batch variation and therefore each batch has to be re-optimized. Tandem dyes are highly variable between different suppliers.
Fluorophore Fluorescence color Maximum excitation (nm) Maximum emission (nm)
Spectrally similar dyes
PE-Alexa Fluor 647   496, 546 667 4  
PE-Cy5   496, 546 667 5  
PE-Cy5.5   496, 546 695 4  
PE-Alexa Fluor 700 Infrared 496, 546 723 2 PE-Cy5, PerCP
PE-Alexa Fluor 750 Infrared 496, 546 779 4 PE-Cy7
APC-Alexa Fluor 750 Infrared 650 779 4 APC-Cy7, DyLight 750
PE-Cy7 Infrared 496, 546 785 4 PE-Alexa Fluor 750
APC-Cy7 Infrared 650 785 2  

Abbreviations: APC; allophycocyanin, FITC; fluorescein isothiocyanate, PE; phycoerythrin (Note. phycoerythrin (PE) is the same as R-phycoerythrin (RPE)), PerCP; peridinin-chlorophyll-protein complex.

Relative Brightness

Although some fluorophores may have similar excitation and emission wavelengths, their relative brightness may be different. Fluorophore brightness depends on how many photons a fluorophore emits when being excited by a laser, as well as the conversion rate of those photons when they hit the detectors and are converted into electrons. The relative brightness can be an important factor in flow cytometry to obtain good signal resolution. If an antigen is highly abundant, most fluorophores can be used, however low abundance antigens and rare cell populations will require bright fluorophores to achieve sufficient separation from the negative population. When building larger panels, good separation will improve the analysis of your data and may allow you some leeway in your fluorophore choices. The graphs below show how fluorophore brightness can affect the appearance of flow cytometry data.

  Laser line Maximum excitation (nm) Maximum emission (nm) Filter Relative brightness
405 410 455 460/22 1
640 702 723 720/60 2
488 490 525 525/35 3
640 650 665 670/30 4
561 546 578 577/15 5

Fig. 1. Fluorophore relative brightness. Examples of staining showing the relative brightness of common fluorophores on the same cell population. Data collected on the ZE5™ Cell Analyzer.

Many factors will affect the appearance of data. This can be the flow cytometer, filters and laser used to excite the fluorophore, the laser power and instrument settings as well as the type of plot chosen. Other examples include the sample used, antibody clone, the F:P ratio and staining protocol. Finally, remember that each fluorophore will influence another in a multicolor panel so compensation and fluorescence spread should be considered when choosing a fluorophore.

For more information about fluorophores and immunophenotyping, refer to our flow cytometry resources.

Click on the links below to find more in-depth information on each topic and view our popular flow cytometry basics guide

Flow Cytometry Resources