Flow Cytometry

Which Fluorophores are Useful for
Flow Cytometry?

There are many fluorescent molecules (fluorophores) with a potential application in flow cytometry. The list is ever growing and we will not cover all of them here. The fluorophores currently available from bio-rad-antibodies.com are described in Tables 1, 2 and 3. With new fluorophores constantly being added, such as new Starbright Dyes, don’t forget to check out our latest flow cytometry products.

Single Dyes

Single dyes such as FITC, PE, APC and PerCP have been available for many years, but there are now alternatives available from Alexa Fluor dyes, which offer users greater photostability and brighter fluorescence. In addition alternative laser lines are becoming more affordable so dyes excited by 355 nm and 405 nm lasers are increasing the options for multiplexing.

Starbright Dyes

StarBright Dyes are novel proprietary fluorescent nanoparticles specifically developed for flow cytometry. They are bright, photostable, have narrow excitation and emission profiles and do not require a special buffer when multiplexing. With 7 dyes already available for the 488 nm and 405 nm lasers and more to be launched later this year, these novel dyes give you more choice and flexibilty when building multicolor panels. Find out more about Starbright Dyes.

Tandem Dyes

Tandem dyes comprise a small fluorophore covalently coupled to another fluorophore. When the first dye is excited and reaches its maximal excited electronic singlet state, its energy is transferred to the second dye (an acceptor molecule). This activates the second fluorophore, which then produces the fluorescence emission. The process is called Förster resonance energy transfer (FRET). It is a clever way to achieve a higher Stokes shift and therefore increase the number of colors that can be analyzed from a single laser wavelength.

The majority of tandem dyes have been manufactured for the 488 nm and 640 nm lasers which are found in most cytometers. Tandem dyes are very useful for multicolor fluorescence studies, especially in combination with single dyes. For example Alexa Fluor 488, phycoerythrin (PE), peridinin chlorophyll protein (PerCP)-Cy5.5 and PE-Texas Red can all be excited at 488 nm, but will produce green, yellow, red and infrared emissions, respectively, which can then be measured using separate detectors.

Fluorescent Proteins

Fluorescent proteins, such as green fluorescent protein (GFP), have become an integral tool for understanding protein expression in many scientific disciplines. Other fluorescent proteins, such as mCherry and yellow fluorescent protein, have also become widely used for flow cytometry analysis and cell sorting. The fluorescent proteins are often co-expressed or expressed as a fusion with the protein of interest. The benefit of these fluorescent proteins is the quantitation of intracellular markers in live cells without requiring permeabilization of the cell membrane. Common fluorescent proteins are listed in Table 4.


Table 1. Fluorophores for flow cytometry.

Fluorophores Fluorescence Color Maximum Excitation (nm) Maximal Emission (nm) Relative Brightness
DyLight 405   400 420 3
Alexa Fluor 405   401 421 3
Pacific Blue   410 455 1
Alexa Fluor 488   495 519 3
FITC   490 525 3
DyLight 550   562 576 4
PE*   490; 565 578 5
APC   650 661 4
Alexa Fluor 647   650 665 4
DyLight 650   654 673 4
PerCP   490 675 2
Alexa Fluor 700 Infrared 702 723 2

* PE is the same as R-phycoerythrin.
APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridinin chlorophyll protein.


Table 2. StarBright Dyes.

Fluorophores Fluorescence Color Maximum Excitation (nm) Maximum Emission (nm) Relative Brightness
StarBright Violet 440   383 436 5
StarBright Violet 515   402 516 5
StarBright Violet 610   402 607 5
StarBright Violet 670   401 667 5
StarBright Blue 700   470 705 5

Table 3. Tandem dyes for flow cytometry.

Fluorophores Fluorescence Color Maximum Excitation (nm) Maximal Emission (nm) Relative Brightness
PE-Alexa Fluor® 647   496, 546 667 4
PE-Cy5   496, 546 667 5
PerCP-Cy5.5   490 695 3
PE-Cy5.5   496, 546 695 4
PE-Alexa Fluor® 750 Infrared 496, 546 779 4
PE-Cy7 Infrared 496, 546 785 2
APC-Cy7   650 785 2

* PE is the same as R-phycoerythrin.
APC, allophycocyanin; PE, phycoerythrin; PerCP, peridinin chlorophyll protein.


Table 4. Fluorescent proteins in flow cytometry.

Fluorophores Fluorescence Color Maximum Excitation (nm) Maximal Emission (nm) Relative Brightness
EGFP   383 445 2
CFP   439 476 2
EGFP   484 509 4
YFP   514 527 5
RFP   558 583 4
mCHERRY   587 610 3