Biomarkers supply scientists with tools to unravel and battle diseases. With so many diseases and even more potential biomarkers, scientists need automated methods to find and work with these biomarkers, for example, meaningful molecular markers on a cell’s surface. For such work, researchers often turn to fluorescence-activated cell sorting (FACS), which is a form of flow cytometry.
FACS sorts a mixture of cells—one by one—into specific groups based on features detected by measuring a fluorescence tag. The late Leonard Herzenberg, a Stanford University geneticist, developed the first FACS device, which earned him the Kyoto Prize in 2006. Although he won that prize, he insisted on acknowledging the contribution from Leonore Herzenberg, who was his wife and remains a professor of genetics at Stanford. As the Herzenbergs built this technology through teamwork, so it has gone on to influence, if not drive, the work of many other scientists.
In fact, a recent search of FACS on PubMed.gov returned more than 12,000 publications.
The speed and specificity of FACS makes it a crucial tool in cell biology. As Robert Balderas, vice president, market development, at BD Biosciences (Becton, Dickinson and Company), explains, “FACS is used for cell sorting, and it is also applied to analyze a cell, such as discovering biomarkers on cells.”
Super-charged sorting
Simply put, FACS sorts cells based on a dye attached to an affinity reagent. In most cases, an antibody serves as that reagent, and it binds something on or even inside a cell. “Flow is used to measure what’s on a cell, in a cell or made by a cell,” Balderas summarizes.
A FACS platform measures and reacts to the emission from the dye, and more than one dye can be used in the same experiment. Consider the use of two dyes that bind to different targets. When a cell runs through a FACS, it gets placed in one of four categories: positive for one dye, positive for the other dye, positive for both dyes or negative for both dyes. So, the number of possibilities is 2n, where n is the number of dyes. “Most of the research uses up to about 20 parameters,” Balderas says. That many dyes create more than 1 million possible combinations.
The brightness of the dye, however, limits the possible distinctions.
The required brightness of the dye is inversely related to the prevalence of the target.
So a brighter dye is needed for a lower-abundance target, and a less-bright dye can be used on a higher-abundance target.
Also, different components can be used to create the signal. In 2011, Garry Nolan, a Stanford professor of microbiology and immunology and part of the Herzenberg academic family tree, and colleagues, including Balderas, used antibodies, metal-encoded DNA and other indicators to measure 34 parameters [1]. That platform divided cells into more than 17 billion categories. This technique, wrote the authors, provided a “systems-level view of human hematopoiesis and immunology from the perspective of immunophenotype and coupled it to underlying events as measured through receptor engagement and small-molecule drug actions.”
Biocompare’s Cell Sorters Search Tool
Find, compare and review Cell Sorters
from different suppliers Search
The capabilities keep increasing. For example, BD Biosciences recently developed a platform (BD FACSymphony) that can accommodate 50 parameters. That pushes the possible categories beyond a quadrillion, or a million billion. Pushing this platform up to just 40 parameters—the most used in practice so far—provides more than one trillion categories. “With 40 different reagents with different colors and looking at all the different cells that might be in, say, blood, we look deeper and deeper into biology,” Balderas says.
Exploring exosomes
Some cells secrete small vesicles called exosomes, and they play roles in many functions, such as coagulating blood. Despite the growing interest in studying exosomes, it can be difficult to do. “In the field of exosome research, traditional methods like Western blot or electron microscopy lack throughput or require large amounts of sample to study expression of antigens on exosome populations,” says Johannes Fleischer, product manager for flow cytometry at Miltenyi Biotec. “Flow cytometry has become an increasingly popular tool for exosome analysis in the past years, as it allows fast and high-throughput analysis of exosome samples and thus facilitates biomarker discovery.”
To help researchers work with these vesicles, Miltenyi Biotec created its MACSplex Exosome Analysis Kit. This bead-based flow-cytometry assay can simultaneously analyze up to 37 antigens on exosomes. “By this method,” says Fleischer, “researchers can easily identify different antigen expression between treated/untreated or healthy/disease conditions.”
A research team from Miltenyi Biotec and colleagues studied the effect of melanoma on the composition of exosomes in patients’ plasma [2]. These vesicles can be used as diagnostics in melanoma, and they might be involved in tumor progression. The authors stated that their results “argue for an indirect influence of melanoma cells on the vesicle secretion or vesicle protein loading by blood cells.”
Increasing the capabilities
At the Johns Hopkins School of Medicine, associate professor of physiology Dax Fu and his colleagues focus FACS on disease-related biomarkers. One of these is zinc transporter 8 (ZnT8) on pancreatic β cells, which make insulin, which in turn plays a role in regulating glucose levels in the blood. That function connects ZnT8 to diabetes.
Fu explains: “We inserted a peptide tag to the extracellular surface of ZnT8, used anti-tag antibodies to label the protein on cell surface and then used a fluorescent secondary antibody for FACS and quantification of ZnT8 surfacing as a function of glucose-stimulated insulin secretion, a hallmark function of pancreatic beta-cells” [3].
This technique shows that ZnT8 can be used to track and purify pancreatic β cells. Fu adds, “We are developing specific anti-ZnT8 monoclonal antibodies directed to native epitopes on the extracellular surface of beta-cells that will be used for: imaging beta-cells in pancreatic tissues; purifying functional beta-cells from pancreatic progenitor cells; and high-throughput screening of antidiabetogenic drugs using ZnT8 surface display as a functional readout.”
As scientists push FACS further, they desire an increasing selection of features.
For example, Jason McCormick, manager of flow cytometry at Weill Cornell Medicine, says he would like “better instrumentation in terms of spectral detection range and sensitivity, better reagents in terms of fluorochrome excitation/emission properties and better availability of marker-fluorochrome combinations—and we are quickly getting there.”
The teamwork between research scientists, instrument manufacturers and dye developers drives FACS forward. As Balderas says, “The field of flow cytometry is a great example of cocreation.”
References
[1] Bendall, SC, et al., “Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum,” Science, 332:687-696, 2011. [PMID: 21551058]
[2] Koliha, N, et al., “Melanoma affects the composition of blood cell-derived extracellular vesicles,” Frontiers in Immunology, 7:282, 2016. [PMID: 27507971]
[3] Huang, Q, et al., “Coupling of insulin secretion and display of a granule-resident Zinc transporter ZnT8 on the surface of pancreatic beta-cells,” J Biol Chem [epub ahead of print], 2017. [PMID: 28130446]
Image: Shutterstock Images