Flow Cytometry Improvements Drive Research Advances

By fostering a close relationship with the life sciences and flow cytometry communities, instrument and reagent manufacturers are helping to drive advances in research, healthcare, and technology. Here, we look at some recent developments and consider the rationale behind them.

Unmet demands and challenges

According to Mike Blundell Ph.D., Product Manager at Bio-Rad Laboratories, flow cytometry can now achieve more than most researchers generally require. “For simple immunophenotyping, most panels fall between 6 to 15 colors,” he says. “However, conventional instruments can allow you to create panels with over 25 colors, and in full-spectrum flow cytometry this is increased to over 40, provided the right antibodies are available conjugated to the right fluorescent dyes.” Ultimately, whether the existing capabilities of flow cytometry are sufficient to meet your needs largely comes down to the aims of your research.

“There are certainly still unmet demands and challenges for flow cytometry,” reports Maria C. Jaimes, VP of Scientific Commercialization at Cytek^® Biosciences. “For example, ease-of-use and standardization are areas in which researchers would like to see more improvements. Also, for those running highly multiparametric panels, efficient and automated analysis tools are still limited. The growing expectation in the industry is that, in the future, analysis workflows will be fully automated. In addition, options that effectively combine large datasets from different technologies, such as flow cytometry and RNA-seq, are desirable.”

Are you capitalizing on flow cytometry advances?

It can be easy to get stuck in a rut when running flow cytometry experiments, not least because redesigning panels takes time. But there are advantages to be gained by scrutinizing how your research is being performed. Kenta Yamamoto, Product Manager for Cell Analysis at BioLegend (now part of Revvity), suggests considering the following:

• Are you routinely combining data from several small flow cytometry panels?

Think about re-visiting the capabilities of your flow cytometer and condensing to fewer, but larger, multicolor panels. Higher multiplexing enables deeper characterization of your samples by allowing for co-expression analysis across a broader number of markers simultaneously.

• Are you using fluorophore and marker combinations left behind by lab predecessors many years ago?

Speak with vendors who are innovating with new flow cytometry reagents to see whether you can make any adjustments to acquire better data. This could include swapping out dimmer fluorophores for new, brighter alternatives to better stain scarce target cell populations, or using fluorophores that minimize spectral overlap to simplify analysis.

Flow cytometry is now faster, easier, and more powerful

As well as enabling researchers to detect more markers, modern flow cytometry offers many other advantages. “Over the years we’ve seen significant improvements in instrument size,” notes Garret Guenther, Ph.D., Senior Product Manager for Flow Cytometry within the Cell Analysis Division at Agilent Technologies. “Specifically, the need to have a dedicated room for a single instrument has been removed, as the advanced instruments of today can fit on a lab bench. There have also been improvements in sensitivity and data quality, both from the hardware and software sides, and in ease-of-use. Other notable benefits of modern systems include faster acquisition times, the ability to detect smaller particles, and the option to increase throughput with auto sampling or robotic automation.”

Advances in flow cytometry instrumentation have been complemented by advances in fluorescent dyes. "Recently, dye improvements have included tunable brightness and spectral properties, which let researchers create larger panels with reduced spillover and spreading,” says Blundell. “Spillover and spreading can especially impact the resolution of rare and low antigen density populations, so having greater control over dye characteristics can improve researchers' chances of accurately detecting these cell types." Dyes have also been developed to exhibit improved stability, lot-to-lot reproducibility, and spectral consistency, and to address common experimental challenges. For example, Bio-Rad’s StarBright Dyes are compatible with a broad range of buffers and can be premixed to save time and reduce error, while BioLegend’s Fire and Spark Dyes can fill previously unused spectral spaces to maximize panel flexibility.

Another important breakthrough has been the development of high-resolution spectral cytometers such as the Cytek® Aurora and Cytek® Northern Lights™. “Spectral cytometers differ from conventional flow cytometers in that they collect all of the light emitted by each fluorophore rather than just a discreet range of wavelengths,” explains Gil Reinin, Senior Director of Marketing at Cytek^® Biosciences. “This allows for the use of spectrally overlapping dyes to significantly increase the number of colors that can be combined in a single experiment.” Cytek^® Biosciences has also recently acquired the Flow Cytometry and Imaging business unit from Luminex Corporation to offer the Cytek^® Amnis^® imaging flow cytometers, which pair flow cytometry with high-throughput imaging capabilities. “With imaging flow cytometry, researchers not only obtain insights about the phenotype of their cells, but also about the morphology and localization of intracellular molecules,” comments Reinin.

Advances in flow cytometry are driving discovery

Advances in flow cytometry have led to countless scientific breakthroughs. The following are just a few recent examples:

• In 2018, Dunker et al. combined high-throughput cell imaging flow cytometry with deep learning to identify and enumerate phytoplankton species, a critical step during water quality assessment that has historically been time-consuming or inaccurate
• In 2021, Samanovic et al. used high-dimensional spectral flow cytometry to characterize cellular and humoral responses to the BNT162b2 (Pfizer BioNTech) SARS-CoV-2 vaccine in uninfected and previously infected individuals, and showed that immunological history may be important for vaccine and booster regimens
• In 2022, one of the latest OMIPs to be published (OMIP-086) described a 25-parameter, 22-color full spectrum flow cytometry panel for the comprehensive enumeration and functional characterization of innate lymphoid cell subsets in mouse tissues

Collaboration between researchers and manufacturers is vital

To ensure that researchers have the necessary tools to answer increasingly complex scientific questions, open communication between end users and manufacturers is vital. “Our field application scientists and technical service team are our biggest assets in understanding researchers’ needs,” says Yamamoto. “Through exchanges with our customers, we are able to gain a clear picture of the current flow cytometry market, including what researchers are working on and any gaps that we can potentially fill with new products.” Guenther echoes Yamamoto’s point about working directly with researchers and also adds that by looking at flow cytometry market trends and continually monitoring publications for new applications, advancements, and discoveries, Agilent is able to develop instruments and reagents that align closely with researchers’ requirements.

Conferences and other scientific gatherings are also useful to guide future development. “Our technical applications and sales specialists organize regular user group meetings to both interact with our customers in a manner specific to our flow cytometry solutions and allow our users to learn from each other,” says Jaimes. “We also monitor the Purdue Flow Cytometry List, which is another great source of information and an invaluable tool for the flow community.”

Lastly, Blundell points out that it is critical for flow cytometry companies to remain customer focused. “Finding out what customers want, either to improve their flow cytometry experiments or to achieve something that is not currently possible with existing tools and technologies, is fundamental to shape future advances in flow cytometry,” he says. “Developing the ‘next big thing’ is all well and good, but such developments can only be considered truly valuable if they deliver on researchers’ expectations.”