laser to minimize potential cross-excitation of the violet dyes. BUV
dyes have progressively changed flow cytometry while addressing
several unmet needs for the scientific community.
Brilliant dyes help build brilliant panels
Before the development of BUV dyes, many 5-laser instruments
came equipped with a 375-nm ultraviolet (UV) laser in addition to
the four common violet, blue, yellow-green (YG) and red lasers.
However, scientists used the UV laser almost exclusively to detect
DNA-binding dyes such as DAPI and Hoechst in cell viability and
cell cycle studies or to detect hematopoietic side populations. This
configuration enabled detection of 16 phenotypic markers plus two
functional dyes, but moving beyond that was difficult.
Scientists who need to design complex multicolor panels are
often forcibly guided by reagent availability limitations rather than
proper panel design practices. The first two BUV dyes (BUV395
and BUV737) conjugated to monoclonal antibodies increased the
choice of available reagents while still enabling the simultaneous
detection of 18 phenotypic markers. However, designing 18-color
panels remained challenging, especially because of visible spectrum
crowding (six violet detectors and five YG detectors) and the use of
high spillover fluorochromes such as PE-Cy5 and PE-Cy5.5.
The development of two additional BUV dyes (BUV563 and BUV661)
did not increase the number of detectable parameters but rather
offered an alternative to PE-Cy5 and PE-Cy5.5, easing visible
spectrum crowding, providing less spillover and enabling a more
balanced distribution of fluorochromes across the five lasers.
The addition of three more dyes (BUV496, BUV615 and BUV805)
to the BUV dye family, together with the development of higher
capability flow cytometers able to detect up to 50 parameters,
eventually allowed scientists to break the 18-parameter barrier.
This opened the way to high-parameter flow cytometry, enabling,
for the first time, the detection of up to 28 colors via conventional
flow cytometry when BUV dyes were used in combination with other
new dyes. The exponential and sudden increase in resolution power
enabled cell characterization at an unprecedented depth. Moreover,
these three dyes also facilitated a more balanced fluorochrome
distribution across the five lasers (Figure 1). The resulting reduction
in spillover not only made complex panels possible, it also simplified
the process of designing less complex panels.
To further complement the BUV dyes and provide customers with
panel design flexibility and expanded flow cytometry capabilities,
the scientists at BD created BD OptiBuildTM Reagents, leveraging a
groundbreaking technology to produce on-demand conjugations. This
resulted in the rapid expansion of BUV dye-conjugated reagents and
products to support the community’s ever-evolving research needs.Making UV mainstream
The rapid expansion of both the BUV fluorochromes and the BUV
reagent portfolio established the UV laser as indispensable for high-
parameter flow cytometry panels run using instruments such as BD
FACSymphonyTM A3 and A5 Cell Analyzers and the BD FACSymphonyTM
S6 Cell Sorter. Today, any high-parameter conventional flow cytometer
on the market is equipped with an UV laser and relies on BUV dyes.The ultraviolet laser and BUV dyes also play a critical role in the
development and adoption of spectral flow cytometry today, enabling
the simultaneous detection of over 40 parameters. With the evolving
advancement of spectral flow cytometry, there are clear innovation
opportunities in the BUV dyes (Figure 2) and BD Biosciences is
uniquely positioned to continue to lead the dye revolution with its line
of BD Horizon Brilliant™ Ultraviolet and BD OptiBuild™ Reagents.(^0300)
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Conventional Flow Cytometry
Spectral Flow Cytometry
Conceptual Rendering
For more information on the UV laser, BUV dyes, and the increased depth of biology that they provide for both conventional and spectral flow cytometry, please visit
bdbiosciences.com/buv
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD, FACSymphony, Horizon Brilliant, Horizon Brilliant Violet and OptiBuild are trademarks of Becton, Dickinson and Company or its affiliates. Cy is a trademark of Global Life Sciences Solutions Germany GmbH
or an affiliate doing business as Cytiva. All other trademarks are the property of their respective owners. © 2021 BD. All rights reserved. BD-25934 (v1.0) 0221
Figure 2: The number of detectable fluorochromes per laser in conventional flow
cytometry is limited by the capture of the emission peak. Spectral flow cytometry
distinguishes fluorochromes based on full spectrum signatures, thus enabling detection
of more fluorochromes per laser.