Flow cytometry antibodies, kits, reagents

Chapter 2: Principles of Fluorescence

Which Fluorophores are Useful for Flow Cytometry?

There are many of fluorescent molecules (fluorophores) with a potential application in flow cytometry. The list is ever growing, but it is not the scope of this applications guide to cover them all. Some of the most useful fluorophores for surface or intracellular epitope detection are described in Tables 1 and 2. There is enough variation in the two tables to cover most researchers’ needs.

Single and Tandem Dyes

Single dyes such, as FITC, PE, APC, and PerCP have been the standard for many years but are now facing competition from alternatives like Alexa Fluor Dyes, which offer the user greater photostability and brighter fluorescence.

Tandem dyes comprise a small fluorophore covalently coupled to another, larger 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 fluorescence 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 standard 488 nm laser, which is found in most flow 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 be measured using separate detectors.

Fluorescent Proteins

Fluorescent proteins (FPs), 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 eYFP, have also become widely used for flow cytometry analysis and cell sorting.

Often, FPs are co-expressed with or directly conjugated to the protein of interest. The use of FPs allows the quantitation of intracellular markers without requiring permeabilization of the cell membrane.

Table 1. Fluorophores for flow cytometry.

Fluorophores Fluorescence color Maximal absorbance, nm Maximal emission, nm Relative brightness
DyLight 405   400 420 3
Alexa Fluor 405   401 421 3
Pacific Blue   410 455 1
DyLight 488   493 518 4
Alexa Fluor 488   495 519 3
FITC   490 525 3
DyLight 550   562 576 4
PE   490; 565 578 5
Texas Red   596 615 2
APC   650 661 4
Alexa Fluor 647   650 665 4
Cy5   649 670 3
DyLight 650   654 673 4
PerCP   490 675 2
DyLight 680   692 712 4
Alexa Fluor 700 Infrared 702 723 2
DyLight 755 Infrared 752 778 4
DyLight 800 Infrared 777 794 4

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

Table 2. Tandem dyes for flow cytometry

Fluorophores Fluorescence color Maximal absorbance, nm Maximal emission, nm Relative brightness
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 Infrar ed 496, 546 723 2
PE-Alexa Fluor® 750 Infrar ed 496, 546 779 4
APC-Alexa Fluor® 750 Infrared 650 779 4
PE-Cy7 Infrar ed 496, 546 785 2
APC-Cy7 Infrared 650 785 2

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


Alexa Fluor® and Pacific Blue™ are trademarks of Molecular Probes Inc, USA
DyLight® is a trademark of Thermo Fisher Scientific and its subsidiaries