New methods in high-parameter flow cytometry are opening research frontiers for scientists, namely multi-dimensional, single-cell profiling experiments.Credit: E+/Getty
Housed inside BD Biosciences’ latest cell sorter, the BD FACSymphony™ S6 Cell Sorter*, is an array of sophisticated optical technologies. There is space for up to 9 different lasers, along with photodiodes for forward scatter and photomultipliers capable of fluorescent and side scatter detection, resulting in an instrument that is configurable up to 50 parameters. Integrated with these lasers are adjustable fluidic arrays, variable sized nozzles, bubble detectors, and a high-speed single cell deposition unit designed to sort purified cell populations into tubes, slides or plates ranging from 6- to 384-wells. Sample loading is automated, drawing from microtubes, 12x75-mm tubes, or 15-mL tubes, all under constant agitation, if required, and with temperature control.
These features enable more than 25 colours to phenotypically define and isolate distinct cell populations. Welcome to the world of high-dimensional biology, where researchers are figuring out new ways to study the functions of individual cells.
The road to multiplicity
“The history of flow cytometry is a magnificent story of science and technology coming together,” says Robert Balderas, vice president of biological sciences at BD Biosciences.
Modern flow cytometry dates back to the early 1970s when a team at Stanford University, led by Leonard Herzenberg, merged antibodies tagged with fluorescent labels with cell sorting and fluorescence microscopy to build the first fluorescence-activated cell sorter.1 Shortly thereafter, a commercial flow cytometer was introduced by Becton Dickinson, based on Herzenberg’s work, and the use of flow cytometry blossomed as scientists embraced the technique to sort and isolate individual cells.
While flow cytometry immediately impacted the fields of immunology, virology, and cancer biology by providing insights into different cellular phenotypes and functions, by the late 1990s researchers began understanding the importance of studying single cells from heterogenous populations within complex biological environments. However, such experiments were problematic for flow cytometry as there weren’t enough different fluorophores available to identify multiple cell phenotypes at the time. Researchers turned their attention to mass spectrometry techniques, and later single cell RNA sequencing (scRNA-seq), to fill the voids.
Sidelined, flow cytometry would find its footing through the work of researchers such as the late Roger Tsien from the University of California, San Diego, and others, who developed ways to engineer fluorescent proteins with different excitation and emission properties. These efforts, along with the advent of new dyes that could be conjugated to antibodies, led to a continuous growth in the numbers of available fluorophores, reaching more than 100 dyes and fluorescent proteins for use in flow cytometry by 2020. Developers jumped at these advances, designing new instruments with more lasers and detection capabilities, such as the BD FACSymphony™ S6 Cell Sorter described above, to expand the number of parameters analysed in a flow cytometry experiment and move closer to high-dimensional biology.
High-dimensional biology takes the stage
Researchers also took note of this new capacity to isolate single cells in high-throughput. Jenny Jiang’s lab at the University of Pennsylvania studies T cell biology. Several years ago, Jiang and her colleagues became interested in defining the sequences of antigens that bind T cells as well as the receptors they bind in large numbers. For this, Jiang needed to not only sort cells, but also to obtain sequence information from those sorted cells.
Jiang’s approach was to first use flow cytometry to sort T cells that were cross-reactive to both cancer neo-antigens and wildtype antigens. “This way, we could target these cells in our assays,” she explains. From there, her team sorted this population of cells into single cells, so antigen and T cell receptor-sequence information could be obtained using DNA sequencing. They called the method TetTCR-seq, and showed that it could be used to pair large numbers of antigens with their cognate T cell receptors.2
Recently, Jiang and her team expanded the capabilities of TetTCR-seq by integrating gene expression profiling and protein analysis into the work flow, calling the new method TetTCR-SeqHD.3
“TetTCR-SeqHD truly opens the door for multi-dimensional profiling of single T cell and obtaining TCR and antigen sequences at the same time,” says Jiang, who thinks the technique will also prove useful for developing new therapeutics for auto-immune diseases and cancer.
Jiang’s not alone in embracing multi-dimensional profiling approaches in single cell experiments. Jonas Schulte-Schrepping from the University of Bonn, Germany, and colleagues recently described how high-parameter flow cytometry, along with scRNA-seq and cytometry by time of flight, could be used to provide new insights into the systemic immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).4 Another study led by Dorin-Mirel Popescu from the UK’s Newcastle University, published in Nature, used high-parameter flow cytometry to assist in the identification of human blood and immune cells present during development to create a map of fetal liver hematopoiesis.5
Researchers are starting to push current high-parameter flow cytometry platforms in their efforts to explore single cells. In June 2020, John Connolly from the Agency for Science, Technology and Research (A*STAR), Singapore, and colleagues described their technique called Met-Flow which uses a 27-parameter flow cytometry panel focused on metabolic proteins to examine changes in metabolic states of immune cell subpopulations.6 However, the benefits of using this many colours in a flow cytometry panel does not come without challenges.
Reducing complexity
According to Balderas, there is an art and a science to developing high-parameter flow cytometry antibody panels. Mixing and matching up to 28 different fluorophores attached to different antibodies to ensure that there is only limited scatter or emission overlap is tricky. The good news is that a variety of resources have emerged within the flow cytometry community to help.
The journal Cytometry Part A offers a special publication format dedicated to reports on the design and use of optimized multicolour immunofluorescence panels (OMIPs). According to the journal, the goals of OMIPs are to reduce the time needed for researchers to develop similar panels, provide a starting point for creating novel OMIPs, and give credit to the developers of new panels.
Dozens of OMIPs have been published, with recent articles describing the use of up to 28 different colours in high parameter panels. In addition to the availability of OMIPs, many commercial developers now provide online tools to assist in creation of new flow cytometry panels.
Something for everyone
“High-parameter flow cytometry is going to enrich our understanding of biology,” says Balderas, who notes that more data is always what researchers need to understand biology at a deeper level. While he does not think everyone is going to gravitate to performing large-scale, high-dimensional experiments with flow cytometry, these experiments can provide robust data sets for future studies.
Even as the current crop of flow cytometers become faster with greater throughput, and fluorophores and panel design become better, Balderas believes the best is yet to come. “The field of flow cytometry has a rich future.”
*For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product.