Researchers designing flow cytometry panels have become spoiled for choice with regards to fluorophore selection. Well-known dyes such as APC, FITC, and PE are now complemented by newer fluorophores with increased brightness, narrower excitation and emission profiles, and unique spectral signatures. It is even possible to select fluorophores that allow for tuning of dye brightness based on panel requirements. But while the list of available fluorophores continues to grow, it remains essential to consider key dye characteristics during panel design. In this article, we explain why classic dyes retain their appeal and comment on the advantages modern fluorophores bring to flow cytometry-based research.
Pushing the boundaries of multiparameter flow cytometry
Most conventional flow cytometry panels typically contain anywhere between 4–20 colors, with under 10 colors generally being the norm. According to Kenta Yamamoto, product manager for cell analysis at BioLegend, a main reason for this is technical ease of use, since smaller panels reduce the complexity of spillover and instrument compensation considerations. Another reason is the need to repeat panels used in the past, when multiparameter analysis may have been more limited. However, higher parameter flow cytometry has seen significant uptake in recent years. “Early flow cytometers commonly allowed for analysis of around four colors,” he says, “but parameter capabilities are rapidly increasing. State-of-the-art instruments and the availability of fluorophores with novel spectral properties have enabled successful analysis of up to around 40 colors from a sample. These developments are paving the way for more researchers to adopt higher parameter flow to identify unique cell populations and answer more complicated scientific questions.”
Are the old fluorophores still the best?
Many flow cytometry panels still include well-known fluorophores such as APC, FITC, PE, Alexa Fluor dyes, and PE tandems. “These dyes have been available for over 20 years and are popular because they are trusted to work, have an extensive publication history, and are reasonably priced,” reports Mike Blundell, product manager at Bio-Rad. “Moreover, virtually every flow cytometer has the correct lasers and filters required for their detection and analysis. The spectral characteristics of established fluorophores are also well-known, as is how to compensate effectively, and these dyes are available conjugated to a wide range of antibodies so can readily be incorporated into flow cytometry panels.”
For these reasons, researchers can be hesitant to switch to using newer fluorophores for flow cytometry. Yamamoto observes that while some experienced multiparameter flow users may be eager to introduce new fluorophores to a panel to measure more parameters and drill deeper into unique cell sub-sets, other researchers may experience more trepidation. “Using a new fluorophore can mean rearranging an existing panel to fit more colors in, dealing with additional spectral spillover and compensation, and trusting in a reagent that has not yet been widely published in the literature,” he says. “Appropriate instrument availability is also key—not all older instruments are designed to optimally detect novel fluorophores, and vice versa. Identifying which fluorophores best fit both the experiment and the available instrumentation is key to success.”
“The saying ‘if it’s not broken, don’t fix it’ may be applicable when considering including new fluorophores in a flow cytometry panel,” says Eric Torres, Ph.D., marketing manager at Biotium. “Yet new dyes that are spectrally similar to traditional fluorophores can bring many advantages to flow cytometry, even when using older instrumentation. For example, if a new fluorophore is brighter than a current dye, it can be beneficial in an older system that has less sensitive detectors than more recent models. As the performance of new dyes continues to be validated, I’m optimistic that researchers will eventually replace older dyes with newer ones.”
Anson Blanks, Ph.D., customer success scientist at Phitonex, comments that researchers using spectral cytometry are often more open to using novel fluorophores compared to researchers performing conventional flow cytometry experiments because they depend on the availability of new colors to build out their ever-expanding panels. “Advances in spectral cytometry have allowed Phitonex to build a 45-color spectral panel, which is the largest panel to date,” reports Blanks. “We’re also aware of some researchers who are in the process of building 50+ color spectral panels.”
Panel design considerations still apply
Irrespective of whether old or new fluorophores are preferred, the same panel design considerations hold true. These include pairing brighter fluorophores with low abundance antigens, and combining dyes with minimal spectral overlap to minimize spillover into nearby channels. “A further important, but frequently overlooked, fluorophore characteristic is spread,” remarks Blanks. “Spread reduces the resolution of flow cytometry results, which can lead to data loss and inaccurate conclusions. In a larger conventional or spectral panel, it is more important to be spectrally clean than it is to be bright to retain biological resolution of the data.”
Another factor to think about during panel design is fluorophore stability. “Tandem dyes can degrade, either due to photosensitivity or long-term storage, leading to decreased FRET and enhanced signal from the acceptor and donor fluorophores that necessitates increased compensation and can give unreliable data,” explains Blundell. “Additionally, some tandems are sensitive to fixation, while certain fluorophores may interact with one another unless they are diluted in a dedicated staining buffer.” Other dyes can bind non-specifically to certain cell types, as typified by Cy dye binding to monocytes and macrophages; specialized blocking buffers are available that prevent this, but buffer compatibility with other dyes in the same panel must always be confirmed.
Recent advances making panel design more flexible
Those engaged in manufacturing reagents for flow cytometry are under constant pressure from researchers to develop novel fluorophores that increase the scope of multiparametric analysis. “Our Fire™ Dyes are tandem fluorophores that are designed to fill spectral spaces previously unused in conventional flow cytometry,” explains Yamamoto. “Some of them have been specifically developed for spectral cytometry applications, such as APC/Fire™ 810, which is ideally suited to the latest spectral cytometers that have enhanced detection in the 800 nm range compared to older instruments. Our new PE/Fire™ 640 is another useful addition to spectral cytometry panels, where it can be combined with widely adopted fluorophores like PE/Dazzle™ 594 and PE/Cyanine5. We’re also actively growing our range of Spark Dyes; these small synthetic fluorophores have relatively narrow excitation and emission profiles and are stable from signal quenching by various standard fixation and permeabilization buffers.”
Bio-Rad’s newest range of fluorophores, StarBright Dyes, also benefits from narrow excitation and emission characteristics and stability in common staining buffers. “Although StarBright dyes are generated from different monomers, they are not traditional tandem dyes and therefore don’t exhibit the cross-laser excitation seen from the acceptor dyes in tandems,” notes Blundell. “The unique spectral signatures allow for their inclusion in spectral cytometry panels while the exceptional brightness provides better resolution of rare populations and low-density antigens. At the end of last year we added StarBright Violet 440, StarBright Violet 515, StarBright Violet 610, StarBright Violet 670, and StarBright Blue 700 to our portfolio, providing researchers with even greater flexibility in panel design.”
Biotium’s CF® dyes are developed using chemical engineering approaches to resolve long-standing limitations of well-known fluorophores. One such example is the non-specific binding and background fluorescence exhibited by dyes such as Alexa Fluor and Cy dyes. “These particular dyes feature a common chemical modification—sulfonation—to reduce dye aggregation and improve solubility,” explains Torres. “However, the increased negative charge is associated with non-specific binding and background fluorescence. By using pegylation to shield the negatively charged sulfonate groups, we’ve been able to overcome these issues as well as further reducing dye aggregation and improving solubility.” The CF® dye range currently comprises over 30 dyes spanning the visible, far-red, and near-IR spectra, with additional colors in development.
“The recent release of NovaFluors™ represents a game-changer for both conventional and spectral cytometry,” says Blanks. “These are produced using our Phiton™ platform, allowing for their construction in a manner that provides improved stability and reduces the risk of laser cross-excitation compared to other dyes (e.g. the excitation of PE and PE-tandem dyes by both blue and yellow-green lasers). This allows conventional cytometer users to expand their panels by opening detectors that were previously unusable due to laser cross-excitation. NovaFluors also have digital brightness, meaning the brightness can be ‘tuned’ to match antigen density without the need to redesign the entire panel. Finally, a major benefit to both conventional and spectral cytometry users is the clean emission profile of NovaFluors. This reduces spread to improve resolution and support even deeper phenotyping.”