Exploring the microbiome and more with faster and more accurate platforms
“Now you can find very rare cells and do things that were not even feasible until a couple of years ago,” says Andrea Cossarizza, president-elect of the International Society for Advancement of Cytometry (ISAC) and professor at the University of Modena in Italy. Using flow cytometry, researchers can find increasingly rare cells because the technology, says Cossarizza, provides “the possibility to analyze huge amounts of cells in a very short time.” Just a few years ago, flow cytometry handled only a few hundred cells per second, but some of today’s platforms can process 100,000 cells every second.
To get the top-end speed, scientists pay top dollar, but according to Cossarizza, there are even “affordable instruments that analyze 20,000 to 40,000 cells per second.” As an example, Cossarizza mentions the Attune NxT from Thermo Fisher Scientific (Waltham, Mass.), which he uses when working on rare cells.
Some platforms can even align the lasers through a software function. That alone increases the reach of this technology, because it requires less expertise in the instrumentation. Furthermore, some instruments, such as the S3e Cell Sorter from Bio-Rad Laboratories (Hercules, Calif.), used by Cossarizza in some cases, feature automation that simplifies instrument set up and operation, allowing users to walk away and attend to other lab tasks as their sample is being sorted.
Today’s most sophisticated flow-cytometry technology promises to reveal more about the microbiome, such as the human-gut bacterium Enterococcus faecalis. (Image courtesy of U.S. Department of Agriculture.)
Lots of lasers
Also key to identifying rare cells and distinguishing them from other cells in a sample is to use more fluorescently tagged labels, and that requires more lasers and more detectors. “Just a couple of years ago, it was very difficult to use even a few lasers, and now you can use five and even more,” Cossarizza says. Using smaller lasers simplifies the configuration of the optical bench, and their lower energy requirements minimize their contribution to experimental noise.
Complementary to smaller lasers are smaller, more sensitive signal detectors. Most flow cytometers use photomultiplier tubes (PMTs) for signal detection, but these can suffer from low sensitivity. Avalanche photodiodes (APDs)—developed to detect small signals in the telecommunications industry—replace PMTs in Beckman Coulter’s CytoFLEX. “By using avalanche photodiodes instead of conventional photomultiplier tubes, a cytometer can collect and resolve low-level signals better than has been previously possible,” says Jeannine T. Holden, director of scientific affairs, flow cytometry at Beckman Coulter Life Sciences (Miami, Fla.). “APDs also make it easier to build more lasers into an instrument, as the lasers required are much smaller and the APDs themselves are also small.” APDs are also more sensitive than PMTs in the far-red wavelengths. “Now you can go from UV to far-red,” Holden says. “That gives you more space to play in.”
With experience in both diagnostic and translational research settings, Holden’s medical background drives an approach that’s ultimately patient-based. She says, “I tend to look at things from a health perspective, whether diagnosing or treating a disease or prevention—whatever we need to do really from a health standpoint.” Some of the most interesting targets in health research today, the microbiome—the microorganisms that live on or in us—and microparticles like exosomes, require exquisitely sensitive instruments in order to distinguish the relevant signals from experimental noise.
To learn even more, it will be worth applying advanced flow cytometry to the microbiome, because “so much of it isn’t characterized,” Holden explains. “For so long, people only cared about pathogens, but there are many critters that live among us and on us and influence our health.”
Despite the advanced technology in Beckman Coulter’s CytoFLEX, scientists can set it up themselves. (Image courtesy of Beckman Coulter.)
Flow cytometry promises to help scientists unravel the microbiome. A team of scientists from Flinders University in Australia used flow cytometry to measure bacteria and virus-like particles that might cause chronic rhinosinusitis.1 They propose that “flow cytometry can be used as a tool to assess microbial biomass dynamics in sinuses and other anatomical locations where infection can cause disease.”
Sánchez-González et al.2 used flow cytometry to study the impact of microbial metabolites on prostate-cancer cells. The team studied pedunculagin, a compound in walnuts that microorganisms convert to urolithin A (UA), which caused cell death in some cases. As the scientists reported: “Our results indicate a potential role of UA as a chemopreventive agent for prostate cancer.” Holden sees increasingly broader uses of flow cytometry when studying the microbiome, but “we’re still not fully leveraging the microbiome,” she says. “A lot of the people who are studying it with flow cytometry are using fairly low-end instruments.” She adds, “Most of the published studies use small, benchtop flow cytometers, which are good for quick-and-dirty results, but that don’t give you a lot of detail.”
Reaching for the rare
Scientists need flow cytometry to detect and then isolate rare populations of cells. This technology can also enrich a sample of rare cells. “You can sort different types of cells, and then analyze them at different stages of maturation,” Cossarizza explains. “Then, you can study the cells with biochemical methods or maybe gene-expression analysis.” This can be applied to basic research or exploring the mechanism behind various diseases.
For improved analysis, especially of rare cells, scientists need brighter markers, and more of them. With today’s catalog of markers that can be used with flow cytometry, says Robert Balderas, vice president of market development for BD Life Sciences (San Jose, Calif.), “we can now see receptors on the surface that we could not have five years ago.”
The expanded collection of markers also provides more complete data. “We are analyzing more parameters in cell samples,” Balderas says, noting that the new BD FACSymphony High-Speed Cell Analyzer measures 50 characteristics of cells. “So we now explore biology in a much deeper way, and we find subsets of cells that we’ve never seen before,” he says.
Spreading cytometer use
Although flow cytometers served as core-facility instruments for some time, lower prices brought these instruments into individual labs. “At the world immunology meeting in Milan three years ago with maybe 5000 abstracts and presentations, more than 90% of the scientists were using flow cytometry,” says Cossarizza. “They were studying antigens, different cell populations, apoptosis, mitochondrial activity and more.”
Today’s simpler technology is also bringing cytometers to individual labs. “Scientists can install our CytoFLEX platform themselves,” Holden says. “Flow cytometry is much easier to use than it used to be.”
“The next experiment is always the best one. … The next paper is always the best one,” says Cossarizza. “Recently, he and his colleagues reported on using flow cytometry to study inflammation and aging in people positive for HIV, who still live slightly shorter lives than healthy people.3 In part, chronic inflammation and immune activation, likely due to the persistence of the virus in different types of CD4+ T cells, could explain the difference in lifespan. The scientists reported that some HIV-positive patients “experience a sort of premature aging, which affects heavily the quality of life.”
To support such studies and others, scientists need instruments that do more in less time, and that is the direction in which flow cytometry technology is headed.
- Carlson-Jones, J.A.; Paterson, J.S. et al. Enumerating virus-like particles and bacterial populations in the sinuses of chronic rhinosinusitis patients using flow cytometry. PLoS One 2016; doi: 10.1371/journal.pone.0155003.
- Sánchez-González, C.; Ciudad C.J. et al. Urolithin A causes p21 up-regulation in prostate cancer cells. Eur. J. Nutrit. 2016; doi: 10.1007/s00394-015-0924-z.
- Nasi, M.; De Biasi, S. et al. Aging and inflammation in patients with HIV infection. Clin. Exper. Immunol. 2016; doi: 10.1111/cei.12814.
Mike May is a freelance writer and editor living in Florida. He can be reached at [email protected]