Which Fluorophores are Useful for Flow Cytometry?

Author: Mike Blundell | Reviewer: Chloe Fenton

Fluorophores and Viability Dyes for Flow Cytometry Reference Sheet

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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, and fixable, with narrow excitation and emission profiles, and they do not require a special buffer when multiplexing.

Over 30 StarBright Dyes are now available conjugated to highly cited immunophenotyping targets across the ultraviolet, violet, blue, yellow, and red lasers, including new Spectral StarBright Dyes specifically designed with spectral flow in mind, offering you more choice and flexibility when building multicolor panels (Table 2).

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.

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

Table 2. StarBright Dyes.

Table 3. Tandem dyes for flow cytometry.

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

Table 4. Fluorescent proteins in flow cytometry.

Key Considerations for Selecting Fluorophores for Flow Cytometry

When choosing the appropriate fluorophores for your experiment, there are several guidelines to keep in mind.  

Key considerations include knowing your flow cytometer's configuration to determine which fluorophores it can detect, pairing low-antigen-density markers with bright fluorophores and vice versa, minimizing spectral overlap, and accounting for fluorophore properties such as fixability and stability. 

StarBright Dyes effectively meet these needs and more, establishing themselves as high-quality options for flow cytometry applications.