Fluorophores for Multi-Color Flow Cytometry

Selecting fluorophores for a multi-color flow cytometry experiment needn’t be a daunting task. With more fluorophores available than ever before, many designed to overcome common flow cytometry problems, researchers can build multi-color panels that address almost any experimental need.

Early fluorophores have stood the test of time

Fluorophores such as phycoerythrin (PE), allophycocyanin (APC), and fluorescein isothiocyanate (FITC) have been used for decades without major issues, making them trusted reagents for scientific research. “A main reason for the enduring popularity of these fluorophores is that they can be analyzed by most, if not all, flow cytometers with standard configurations,” says Kenta Yamamoto, Ph.D., Product Manager, Cell Analysis at BioLegend. “In addition, there is wide availability of PE, APC, and FITC conjugated antibodies from many commercial vendors, making it more likely for researchers to find a directly conjugated antibody to probe their antigen of interest readily available.”

Mike Blundell, Ph.D., Global Product Manager at Bio-Rad, agrees, noting that both PE and APC played a central role in increasing the multiplex capabilities of flow cytometry. “Three or four color flow cytometry was made possible following the discovery that PE tandems could be used to obtain greater Stokes shifts from the 488 nm laser,” he explains. “Likewise, when the 640 nm laser was introduced, APC tandems further increased experimental plex. While the issues of spillover and tandem breakdown remain persistent causes for complaint, people tend to put up with them as PE and APC are fluorophores they know well.”

New fluorophores address common challenges

According to Alexis Madrid, Ph.D., Assistant Director of Bioscience at Biotium, new fluorophores are constantly being developed to improve performance in terms of signal-to-noise and biocompatibility. As an example, she reports that while many commercially available synthetic dyes are heavily sulfonated to improve solubility (by increasing the negative charge), sulfonation can also cause unwanted background fluorescence as a result of dye binding to positively charged moieties. “To circumvent this issue, Biotium uses PEG modification to shield the negatively charged sulfonate groups on certain CF^® Dyes,” she says. “By combining the advantages of sulfonation and pegylation, we have been able to improve solubility and reduce background fluorescence, as well as minimize dye aggregation for higher quantum yields and fluorescence.”

Another way that newer fluorophores are designed to improve on existing products is through increased brightness, although Blundell cautions that this can be a double-edged sword. “Increased brightness allows for better separation of your positive and negative populations, which is particularly important for detection of weakly expressed markers,” he reports. “However, as the brightness increases, so does spillover and the spreading of populations after compensation. When using multiple fluorophores, it is often beneficial to rank both the markers and the fluorophores during panel design to identify effective pairings.”

Yamamoto comments that by improving fluorophore stability under common experimental conditions, manufacturers are promoting versatility of use. For example, fluorophores that can withstand different fixative solutions promise easier introduction into an existing panel. “Being able to include a new fluorophore in a multiplexed flow cytometry experiment without significantly altering existing panels and/or experimental protocols is desirable since it can save researchers’ time,” he says. “Technical service teams are a good resource for general assistance with panel design and can describe key fluorophore properties that should be considered for each experiment.”

Top ten tips for fluorophore selection

• Understand the capabilities of your flow cytometer, including which lasers, filters, and detectors it is equipped with
• Clarify the aims of your experiment, including which different cell types you wish to detect, the number of markers required for accurate identification, and whether marker expression is extracellular or intracellular
• Rank markers and fluorophores to identify effective pairings; match low antigen density markers with bright fluorophores and high antigen density markers with dimmer fluorophores
• When detecting rarer markers, with fewer commercial antibody conjugate options available, consider assigning fluorophores to these markers first for greater flexibility when designing the remainder of the panel
• Choose fluorophores with narrow excitation and emission characteristics where possible, especially when designing larger panels
• Assign fluorophores with high spillover to markers that are mutually exclusive (i.e., markers that are on different cell types, rather than different markers on the same cell)
• Be aware that some fluorophores can bind non-specifically to certain cell types; consider using alternative fluorophores where this is a known problem
• For longitudinal studies, think about purchasing fluorophore-conjugated antibodies in bulk for improved experimental consistency
• Leverage online tools like Biocompare’s Flow Cytometry Panel Builder to simplify panel design
• Speak with vendors for guidance on fluorophore selection

Enhancing the capabilities of flow cytometry

A shared goal of all three companies included here is to push the boundaries of multicolor flow cytometry by developing spectrally unique fluorophores. “BioLegend’s introduction of the Brilliant Violet™ fluorophore family in 2011 allowed researchers to create more advanced multicolor panels with very bright fluorophore options off of the violet laser, which was underutilized at the time,” reports Yamamoto. “More recently, in line with increased use of ultraviolet lasers and the development of spectral flow cytometry, we released Spark UV™ 387 to give researchers even greater flexibility for panel design. We have also broadened our portfolio of Fire tandem dyes, which now includes APC/Fire™ 810 and PE/Fire™ 810, to extend flow cytometry signal detection into the infrared spectrum.”

Bio-Rad’s range of StarBright Dyes, excitable by the 355 nm, 405 nm, and 488 nm lasers, will soon be expanded with the launch of StarBright Yellow. “The StarBright Yellow Dyes have reduced excitation by the 488 nm laser compared to PE and PE-tandems, meaning less compensation is required,” notes Blundell. “Like all of our StarBright products, StarBright Yellow Dyes are extremely bright, have narrow excitation and emission characteristics, and are compatible with any common staining buffer. They also have proven stability for several years with no loss of performance and can be premixed for over 30 days to allow staining of multiple samples over time with the same panel.”

Madrid reports that Biotium carries several unique CF^® tandems, including RPE-CF^®583R and APC-CF^®750T, and is actively developing novel dyes that reach into the far-red and near-infrared (near-IR) range. “Our fluorophore offering includes several near-IR CF^® Dyes with emission above 800 nm which can be excited by the 808 nm laser line,” she says. “Importantly, by developing bright and high-performance dyes in these longer wavelength regions, our aim is to press the flow cytometry instrument companies to utilize these wavelengths by producing the detectors and channels they require. Ultimately, this will further extend the multiplexing capabilities of modern flow cytometry to beyond what is possible today.”