Recent Developments in Flow Cytometry

This article explores what’s new in the flow cytometry space, where novel reagents and instrumentation are expanding flow’s capabilities and making the technology more accessible.

Reagents

Bio-Rad continues to expand its range of StarBright™ Dyes

Bio-Rad’s StarBright Dyes have long been popular for flow cytometry due to their brightness, stability, and narrow excitation and emission characteristics, as well as the ease with which they can be introduced into existing panels. Through its continued expansion of the StarBright Dye range, Bio-Rad offers researchers even greater choice and flexibility when constructing conventional and full-spectrum multicolor flow cytometry panels.

“We completed the StarBright Yellow Dye series in 2023, and the StarBright Red Dye series this year, bringing the total number of StarBright Dyes to 32," reports Mike Blundell, Ph.D., Product Manager at Bio-Rad. “These two series respectively serve as superior alternatives to existing PE and APC dyes and their tandems. For example, the StarBright Yellow Dyes are excited by the 488 nm laser less than PE and can be fixed in PFA and alcohol-based solutions with minimal spectral changes, while the StarBright Red Dyes are also fixable and can be pre-mixed without needing special buffers."

Cell Signaling Technology develops F(ab’)₂ fragment versions of its G4S and Whitlow/218 linker antibodies

The antigen-binding domain of many chimeric antigen receptors (CARs) comprises a single-chain variable fragment (scFv) in which the variable heavy and variable light domains are connected by a G4S linker or a Whitlow/218 linker. In 2023, Cell Signaling Technology (CST^®) received recognition for its anti-G4S and anti-Whitlow/218 linker antibodies, which allow for monitoring the surface expression of virtually any scFv-based CAR using flow cytometry. Now, CST is developing F(ab’)₂ fragment versions of these products to eliminate the problem of antibody binding to Fc receptors. “When using full-length antibodies for studying CAR expression in a mixed population containing monocytes, an Fc blocking step is essential to reduce the likelihood of seeing false positives,” says Chris Manning, Associate Director, Flow Cytometry. “We developed F(ab’)₂ fragment versions of our anti-CAR linker antibodies to eliminate the concern of Fc binding, and shorten protocols with the removal of the Fc blocking step.”

Bio-Techne extends its conjugated antibody offering with mFluor™ Violet Dyes

Bio-Techne is well-known for its extensive portfolio of R&D Systems and Novus Biologicals antibodies, which encompasses around 11,000 products that are validated for flow cytometry. These are available conjugated to almost 30 different dyes, including three mFluor™ Violet Dyes (mFluor Violet 450, 500, and 610) that were added to the company’s commercial offering earlier this year. “The mFluor Violet Dyes are excited by the violet laser (405 nm) and have discrete emission spectra that are readily detected by common flow cytometry channels,” says Emily Cartwright, Ph.D., Product Manager. “Researchers may be especially interested in mFluor Violet 610, which emits in the same channel as Brilliant Violet™ 605, allowing for increased flexibility in flow cytometry panel design. By introducing the mFluor Violet Dyes into our portfolio, we have enabled researchers to do more with Bio-Techne antibodies.”

Abcam’s latest antibody conjugates increase flexibility for intracellular flow cytometry

Finding the right combination of target and label can be challenging. To address this, Abcam has developed an ever-expanding range of over 7,000 fluorescent conjugated antibodies, focusing recently on novel targets for intracellular flow cytometry. “In 2023, we increased the breadth of our conjugation portfolio by launching over 3,000 new combinations of recombinant antibodies and flow labels,” reports Elena Loche, Ph.D., Senior Product Manager, Antibody Conjugation. “Our latest products include conjugated antibodies targeting intracellular molecules, such as cytokines, transcription factors, and phosphorylated proteins, and we are expanding our offering for core metabolic proteins used in Met-Flow for which the range of available conjugates has historically been limited. For even greater flexibility in assay design, we also provide an extensive range of highly reproducible carrier-free, conjugation-ready recombinant antibodies that can be labeled with 40 different fluorophores, including tandem dyes, using our Lightning-Link^® conjugation kits.”

Instrumentation

Beckman Coulter Life Sciences launches the CytoFLEX nano Flow Cytometer

Extracellular vesicles (EVs) are powerful indicators of the biological activity in the cells and systems from which they are released, yet their heterogeneity and small size makes studying them challenging. Launched in March 2024, the CytoFLEX nano Flow Cytometer overcomes known hurdles for EV research with an unparalleled limit of detection by light scatter of particles at least as small as 40 nm relative to NIST traceable polystyrene standards. “The CytoFLEX nano Flow Cytometer is the first purpose-built nanoscale flow cytometer for research use only specifically designed for the analysis of nanoparticles,” explains Matthew Goff, Product Manager. “Up to six simultaneous fluorescent detection channels and five side scatter channels provide the flexibility required to detect smaller EV populations and their low-abundance cargo, while three independent QC tools provide empirical measurements of day-to-day performance.” Other notable features include advanced tools that reduce manual interventions, and improved fluidics that provide >90% accuracy of volumetric measurement and <1% carryover between samples. All of this combines to allow for interrogating 30-50% more target events than conventional cytometry.

BD delivers image-enabled spectral cell sorting with the BD FACSDiscover™ S8 Cell Sorter

“Launched in 2023, the BD FACSDiscover S8 Cell Sorter is the first and only real-time imaging, spectral flow cytometer that enables researchers to sort cells based on real-time spatial and morphological characteristics,” says Dan Moore, Associate Director, Research Software. “By combining next-generation spectral technology with advanced QC systems and robust sorting performance, it delivers easy-to-use high-parameter analysis with the added confidence of images for each cell.” Importantly, the integrated BD CellView™ Image Technology allows for coupling images with traditional flow parameters to sort cells for downstream analysis. “Now, it is possible to perform sorting based on the location of proteins within or around a cell, rather than simply whether the protein is present or not,” comments Moore. “This type of detailed information, which remains hidden in traditional flow cytometry experiments, removes the need for guesswork and lets researchers answer questions that they were previously unable to ask.”

Deepcell provides high-resolution, label-free cell imaging in flow

Being able to monitor changes to cellular morphology in response to various treatments is important to identify potential drug candidates and elucidate mechanisms of action. However, traditional microscopy-based methods are slow, difficult to scale, and dependent on human interpretation. By combining elements of flow cytometry, label-free cell imaging, cell sorting, and artificial intelligence, Deepcell’s REM-I platform represents an entirely new and readily-scalable way to study and sort cells based upon high-dimensional morphological data.

“The REM-I platform takes high-resolution brightfield images of individual cells in flow, with no need for labels or staining,” explains Mahyar Salek, Ph.D., President, Chief Technology Officer, and Co-Founder. “The cells are analyzed by Deepcell’s proprietary deep learning Human Foundation Model, which provides characterization based on 115 dimensions of cell morphology. Because the REM-I platform enables gentle sorting of up to six cell subpopulations that are responding to, or resisting, a particular treatment, it can streamline the drug discovery process.”

Researchers can look forward to further flow cytometry advances as 2024 unfolds—stay tuned to Biocompare for future updates!