To look for specific populations of cells, such as certain types of cancer cells, researchers often combine fluorescent dyes —which bind to molecules on the target cells’ surface and are detected using flow cytometry. To explore more cells or distinguish populations based on more specific surface molecules, scientists enlist dyes that cover a broader spectrum of light, such as the violet and ultraviolet wavelengths. These advances in fluorescent dyes have enabled researchers to perform multiplex target analysis as well as improve signal responses due to reduced background noise and interference. Here we discuss some of the latest fluorescent dyes in the violet and ultraviolet spectrum available for researchers.

Scientists bring flow-cytometry projects involving violet and ultraviolet fluorescent dyes to Stephen Kwok, flow cytometry specialist at the Tufts Laser Cytometry (TLC) core at the Tufts University School of Medicine. Regarding the benefits of such dyes, he says, “For flow cytometry in general, finding the best dye is really a case-by-case process.” He adds, “Some work better on some samples than others, and even the same dye can vary by batch from the same company.” Consequently, Kwok has experienced some variability in the results using different dyes. “One order of a dye might bind a little more or a little less,” he says, “and if it binds more, the output is a little brighter.”

Although Kwok doesn’t think violet and ultraviolet dyes always provide special benefits, he does say that they “give you more options.” In the past, he adds, “People didn’t have those options, or the lasers were harder to get.”

Working at a flow-cytometry core facility, Kwok interacts with scientists using a variety of fluorescent dyes. “People bring in all sorts of stuff,” he says, “and it might be something they are trying the first time, or something that they’ve used forever.”

“Some of the newer dyes across the spectrum are more stable and less prone to quenching,” Kwok says. “Nothing is really far and away better than everything else.”

Building brilliance

Jennifer Wilshire, senior lab manager at STEMCELL Technologies, has used Brilliant dyes from Sirigen. “They have been a real revolution in multicolor flow cytometry,” she says. The Sirigen Brilliant Violet dyes, Wilshire notes, were originally licensed by BioLegend, but then BD Biosciences bought the company. “BD and BioLegend have done well at putting out many, many conjugates with these Brilliant dyes,” Wilshire says. “They have opened up the violet laser in a way that makes it almost necessary to add a violet laser to every flow cytometer.” In fact, she says, “I would not consider buying a cytometer without a violet laser because of these dyes.”

In 2000, American physicist Alan Heeger, New Zealand-born chemist Alan MacDiarmid and Japanese chemist Hideki Shirakawa received the Nobel Prize in Chemistry “for the discovery and development of conductive polymers.” Sirigen patented conducting polymer biosensors based on the Nobel-honored work, which eventually spawned new fluorescent dyes—the Brilliant dyes—that could be used in flow cytometry.

Like other fluorescent dyes, the BD Horizon Brilliant dyes can be attached to antibodies or other biological molecules to target specific surface markers on cells.

Charles Preston Neff , technical director of the allergy and clinical immunology flow cytometry and cell sorting facility at the University of Colorado School of Medicine in Denver, says UV dyes and the BD Horizon Brilliant violet dyes can run in the same flow-cytometry channel and use the same detector. “These dyes are brighter,” he says, “and they give you better differentiation in positive and negative binding.”
As to particular dyes, Neff says BD Horizon Brilliant Violet 421 is “about the brightest.” He adds that Violet 786 is also “really bright,” but that he is not a fan of Violet 510.

The added brightness of these dyes (BD Horizon Brilliant violet dyes) can be a benefit and a challenge.

“You can use more than one marker for various kinds of staining,” Neff explains, “but in some cases you can get overlap, because the signals are so bright.” He adds, “If you use dyes that are a gap away, like 605 and 786, you’re fine.”

Reducing compensation

Lydia Tesfa, assistant operations director of the Einstein Flow Cytometry Laboratory at Albert Einstein College of Medicine, has used a range of dyes in the violet and ultraviolet range for flow cytometry, including Pacific Blue (available from Thermo Fisher Scientific, BD Biosciences, BioLegend and other vendors), BV510 and others. Regarding particular dyes within that range that she likes, she says: “Dyes such as BV605, BV650, BV711, BV786 would be ideal, with of course their matching filters.” One of the benefits of using dyes in this range, says Tesfa, is that “issues with compensations will greatly be minimized.”

Despite the advantages, dyes in this range can create challenges. “Pacific Blue and BV510 are not bright colors,” Tesfa explains, so “compensation can be tricky” between them. Insufficient brightness is not the only issue. “BV421 could sometimes be too bright,” she explains.

Overall, says Tesfa, “The primary challenge is really keeping up with the availability of instrumentation equipped to run these dyes.” As she shares, “Access to a UV laser is not as easy as one would think.”

Deeper density

Different chemistries keep expanding the experimental options available to researchers. For example, “Miltenyi Biotec recently introduced the VioBright technology, which allows an increased number of dye molecules per antibody, as compared to conventional conjugation chemistry,” says Kalpana Singh, product manager, cell analysis, at the company. “The VioBright technology makes dim fluorochromes—such as fluorescein isothiocyanate, or FITC, and FITC analogues—much brighter.”

In most cases, adding too many dye molecules to a biomarker, such as an antibody, eventually quenches the signal.

With the VioBright technology, says Singh, “a much higher number of small dye molecules can be polymerized first and then conjugated to an antibody molecule,” and this prevents signal reduction.

Miltenyi Biotec’s latest two dyes based on this technology are VioBright FITC  and VioBright 515. The latter is based on Vio515, which is a photostable analogue of FITC. “Vio515 is not only photostable but also brighter than the FITC, making VioBright 515 a brighter flurochrome than VioBright FITC,” Singh explains.

In general, tool providers continue to improve upon existing offerings as well as create new ones. Even more related dyes will be available in the future, says Wilshire. “We are excited about the Brilliant UV and Brilliant Blue that are coming.”

The growing list of dyes expands the uses of fluorescent markers tracked with flow cytometry, and scientists can track more targets at once. Multiplexing assays enables researchers to gather more information while potentially using less of their precious samples. The results can provide a deeper understanding of key targets and cellular pathways.

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