The parallel development of instrument hardware and dyes expands the number of total parameters that can be analyzed in a single test run, with each additional parameter leading to an exponential increase in data acquisition capacity and research potential. Thanks to more than 45 years of development, flow cytometry is now an integral protein analysis tool with which scientists can increasingly deepen cellular characterization and profiling.
At the beginning of the new millennium, flow cytometry had reached a plateau as scientists could not collect more than 18 parameters at a time. Around this time Sirigen designed and optimized a novel class of dyes derived from the chemistry behind Alan Heeger, Alan MacDiarmid, and Hideki Shirakawa’s Nobel Prize discovery of conductive polymers. These new polymer-based dyes were groundbreaking, initially leading to a series of direct and energy transfer tandem dyes excited by the violet laser.
BD Biosciences researchers saw the untapped potential of the UV laser and decided to use Sirigen polymer dye technology to create a new family of dyes excited by UV wavelength lasers. Within three years, BD Biosciences developed seven BD Horizon BrilliantTM Ultraviolet (BUV) dyes. These dyes have been designed and optimized to work with the 355 nm UV laser instead of the 375 nm laser to minimize possible cross-excitation of the violet dyes. BUV dyes have gradually transformed flow cytometry while addressing some unmet needs of the scientific community.
Brilliant dyes help build brilliant panels
Before BUV dyes were developed, many 5-laser instruments were equipped with a 375-nm ultraviolet (UV) laser in addition to the four common lasers violet, blue, yellow-green (YG) and red. 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 side hematopoietic populations. This configuration allowed the detection of 16 phenotypic markers plus 2 functional dyes, but it was difficult to go beyond that.
Scientists designing complex, multicolored panels are often forced to be guided by reagent availability constraints rather than proper panel design practices. The first two BUV dyes (BUV395 and BUV737) conjugated to monoclonal antibodies increased the choice of reagents available while allowing the simultaneous detection of 18 phenotypic markers. However, the design of 18-color panels remained challenging, particularly due to the congestion of the visible spectrum (6 violet detectors and 5 YG detectors) and the use of high overflow 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 offered an alternative to PE-Cy5 and PE-Cy5.5, reducing the crowding of the visible spectrum, providing less overflow and allowing more was balanced distribution of the fluorochromes on the five lasers.
The addition of three more dyes (BUV496, BUV615, and BUV805) to the BUV family of dyes, as well as the development of higher-ability flow cytometers that can capture up to 50 parameters, eventually allowed scientists to break the 18-parameter barrier. This opened the way to high-parameter flow cytometry and made it possible for the first time to detect up to 28 colors using conventional flow cytometry when BUV dyes were used in combination with other new dyes. The exponential and sudden increase in resolving power made it possible to characterize cells at unprecedented depths. In addition, these three dyes enabled a more balanced fluorochrome distribution across the five lasers (Figure 1). The resulting reduction in overflow not only made complex panels possible, but also made it easier to design less complex panels.
To further complement the BUV dyes and provide customers with flexibility in panel design and advanced flow cytometry capabilities, BD BD scientists developed OptiBuildTM using a breakthrough technology to create on-demand conjugations. This has resulted in the rapid expansion of BUV dye conjugated reagents and products to meet the ever-evolving research needs of the community.
Make UV mainstream
The rapid expansion of both the BUV fluorochrome and the BUV reagent portfolio has established the UV laser as an indispensable tool for high-parameter flow cytometry panels operated with instruments such as the BD FACSymphonyTM A3 and A5 cell analyzers and the BD FACSymphonyTM S6 cell sorter become. Today, every conventional high parameter flow cytometer on the market is equipped with a UV laser and is based on BUV dyes.
The ultraviolet laser and the BUV dyes also play a crucial role in the development and introduction of spectral flow cytometry and today enable the simultaneous acquisition of over 40 parameters. Looking to the future of flow cytometry, it is clear that BD Biosciences is in a unique position to lead the dye revolution with its line of BD Horizon BrilliantTM Ultraviolet and BD OptiBuildTM reagents (Figure 2).