With the capacity to collect detailed information from millions of individual cells in a matter of seconds, flow cytometry remains one of the most popular laboratory techniques around. The incorporation of specialized fluorescent reagents into staining protocols has enabled researchers to study upwards of 20 parameters within a single sample, making modern-day flow cytometry experiments a far cry from their early predecessors, however this increased complexity is much more involved than simply adding a greater number of labeled antibodies to a sample. To generate accurate and meaningful data, researchers must consider which fluorophores will be used together, which analytes these will target, and whether their instrumentation can detect their chosen readouts effectively.
“The classical use of flow cytometry is to measure forward and side scatter alongside three colors on different lasers,” explains Catherine Klapholz, research scientist at Nanna Therapeutics. “Scatter correlates with cell size and can, for example, allow detection of dead and dying cells, however the choice of colors you use will be dictated by the lasers you have available.”
Stuart Wood, chief technology officer at Nanna Therapeutics, adds that while it was once common practice to use a single laser to excite a selection of dyes exhibiting different Stokes shifts, many research laboratories now choose to use multiple lasers for color separation, especially when setting up more complex flow cytometry panels. “Using different lasers minimizes the amount of work required to compensate for bleed between dye channels and reduces the number of controls you have to run,” he says. “Many high-throughput machines now ship with multiple lasers that are switched on using software, providing greater flexibility to increase panel size, while other instruments have movable filters that let you change the detector set up readily.”
Better availability of antibody-fluorophore conjugates
Wood notes that a significant challenge faced by researchers wishing to set up a multicolor flow cytometry panel is that only a limited number of antibody-fluorophore conjugates are commercially available. “Our experiments are often driven by the reagents we can get hold of and, ideally, the more colors an antibody comes in, the better,” he says. “I would certainly like to see antibody manufacturers routinely offer a choice of conjugates that are compatible with the most common lasers (405, 488, 532/540, and 640), especially since this would make panel design more straightforward, yet many companies don’t provide this option.”
Unfortunately it is often impractical for an antibody manufacturer to supply every antibody in every possible color, although this problem has been addressed by Expedeon, which manufactures easy-to-use kits that allow researchers to label their antibody of choice with an extensive range of fluorescent dyes and proteins. “Lightning-Link® chemistry targets free amine groups,” reports Anna Sereni, head of R&D. “You simply pipette your antibody into a vial of lyophilized reagent, incubate, and then use it in your chosen application. With over 40 different fluorescent labels to choose from, Lightning-Link is ideally suited to multicolor flow cytometry. This is exemplified in a 2014 publication, where eight different antibody conjugates were employed for detailed analysis of oligodendroglial cell populations in murine brains.”
Image: Conjugate Check&Go! kits from Expedeon allow researchers to confirm the success of their antibody conjugation in one easy step using a qualitative lateral flow assay.
“We’re continually expanding the Lightning-Link product range to meet demands for increased panel size,” says Lucy Westbrook-White, head of marketing. “In addition to the highly popular DyLight®, Atto, and Alexa Fluor® labels, we also offer tandem dyes; these represent an excellent way of achieving more readouts from a flow cytometry experiment when the number of excitation lasers becomes a limiting factor.”
Clever strategies to reduce background signal
According to Johannes Fleischer, global product manager at Miltenyi Biotec, flow cytometry panel design is all about getting as much meaning as possible from a flow cytometry experiment. However, as analyses become more complex, researchers must structure panels carefully to ensure data is interpreted correctly.
“Flow cytometry panels easily consist of 10-15 antibodies these days, sometimes making it difficult to distinguish truly positive signals from negative signals within different cell subsets,” says Fleischer. “We’ve addressed this issue in several ways. To overcome the problem of non-specific antibody binding to Fc receptors on immune cells, a major contributor to background signal, we engineered REAfinity™ recombinant antibodies. These have an altered Fc region, eliminating the need for Fc blocking and ensuring reproducible, background-free results. To reduce non-specific binding of tandem dye-conjugated antibodies to monocytes and other non-target cells, a problem flow cytometrists have been aware of for many years, we developed our Tandem Signal Enhancer. We’ve also added VioBright™ dyes to our product portfolio. These are characterized by high fluorescence intensity and fixation stability, making them well-suited to the detection of rare cells, exosomes, or low-expressed surface antigens.”
Fleischer adds that for those wishing to avoid laborious panel design, Miltenyi Biotec offers ready-to-use cocktails such as an 8-color immunophenotyping kit, which contain optimally titrated flow cytometry antibodies and, for added convenience, include accessory products such as isotype controls and dead cell exclusion reagents. The company also has a strong focus on flow cytometry instrumentation and includes the MACSQuant® Analyzer family of flow cytometers, equipped with MACSQuantify™ software for automated calibration and compensation, within their portfolio.
It’s not just about choosing the right fluorophore combination
Stressing the importance of balancing antigen expression levels with fluorophore brightness, Kenta Yamamoto, product manager, flow cytometry at BioLegend, explains that the best method of determining expected cell marker density and cell population frequency within a given sample is to study existing characterization data.
“Utilizing prior knowledge of cell population and marker abundance is useful in setting up a multicolor flow cytometry panel,” he says. “With an ever-growing range of resources available for researchers wishing to set up a multicolor flow cytometry experiment, panel design is much more user-friendly. All of our flow cytometry antibody datasheets include representative staining data that can inform expected expression levels and guide fluorophore selection, and we’ve also developed several tools to guide panel design.”
Included among these tools are BioLegend’s fluorescence spectra analyzer for comparing excitation/emission profiles of different fluorophores; a fluorophore brightness index to provide relative indications of fluorescence intensity; and a multicolor panel selector tool for choosing appropriate fluorophores based upon the instrument set-up and markers of interest. “Taking the time to construct a balanced panel is key to obtaining good flow data,” says Yamamoto, “however one should remember that a well-designed flow experiment is not just about selecting the right combination of fluorophores.”
Biological and technical variability can impact significantly on results and, as with any experiment, researchers must consider other factors for full optimization. “This includes appropriate sample preparation to avoid damaging antigen integrity, titrating antibodies to maximize signal-to-noise ratio, and refining key steps such as fixation, blocking, and washing. It’s also essential that the flow cytometer is in good working order, and that settings such as acquisition gain/voltage, and compensation have been adjusted accordingly using suitable controls.”
With researchers continuing to demand an increasing number of readouts from often-limited sample material, multicolor flow cytometry panels look set to grow further. It’s impossible to guess at the level of complexity they will eventually reach, but it seems fair to say that the evolution of this powerful technique is far from over.