Achieving Reliable Results with Multiparameter Flow Cytometry

Using multiparameter flow cytometry, researchers can now detect upwards of 40 different markers. Here, we look at some of the technological advances making this possible and share tips for achieving reliable results.

Panel size depends on the aim of your research

According to Mike Blundell Ph.D., Global Product Manager at Bio-Rad, the average size of a flow cytometry panel has grown from around 4–6 plex five years ago to 8–10 plex today. But while it is now possible to detect more markers than it was previously, panel size ultimately depends on the biological question you wish to answer. “If you have transfected cells with a fluorescent protein such as GFP, determination of the percent positive is often enough and therefore one marker is sufficient,” he says. “However, if you want to investigate the total immune response to a drug treatment, cell therapy, or vaccine, multiple markers will be needed to detect all of the relevant cell types and come to a rational conclusion.”

Patrick C. Duncker, Ph.D., Cytek U.S. Applications Technical Lead, adds that while a highly focused, hypothesis-driven scientific question may require only 8–14 markers to fully interrogate, a broader, hypothesis-generating immunophenotyping experiment may call for substantially more markers to be used. “The last three years of publications of Optimized Multicolor Immunofluorescence Panels  (OMIPs) show these two paradigms quite clearly, with 7 of 23 panels focused on specific cell subsets using fewer than 14 markers, and the others primarily being immunophenotyping panels using up to 40 markers as seen in OMIP-069. There will always be a place for smaller focused panels, just as there will always be people pushing the limits of what can be analyzed in a single tube. Across both categories, we have seen a general trend toward more markers and more colors in flow cytometry panels.”

Key factors driving increased experimental plex

So, what’s behind this upward shift in panel size? Garret Guenther, Ph.D., Sr. Product Manager at Agilent Technologies, suggests that improvements to flow cytometry instrumentation have played a major role. “The development of flow cytometers with more lasers and more detection channels, such as the NovoCyte Penteon, which can be equipped with up to 5 lasers and as many as 30 fluorescence detection channels, has been fundamental in increasing the number of markers that can be measured simultaneously,” he reports. “In addition, the ability to simplify compensation and experimental setup has facilitated the adoption of these more advanced instruments, as well as helped to improve reproducibility between users and across different labs.”

Notably, the emergence of full-spectrum flow cytometry has made constructing larger panels more achievable. “By utilizing the entire spectrum of light emitted by each fluorochrome, rather than a discrete range of wavelengths, spectral flow cytometers allow researchers to analyze over 40 markers per sample,” explains Duncker. “Not only have the Cytek^® Aurora and Northern Lights™ systems streamlined the panel design process for smaller panels (up to 15 markers on a 3-laser system and 25 markers on a 5-laser system) by providing greater flexibility in fluorochrome choice, but they have also incentivized the development of novel fluorochromes like Cytek’s cFluor^® reagents. For example, cFluor^® YG584 and PE are indistinguishable on a conventional flow cytometer, yet they can be used in combination on a Cytek full-spectrum system, giving researchers another fluorochrome option when building larger flow cytometry panels.”

Echoing Duncker’s point that improvements to both instrumentation and reagents are driving the trend toward building larger panels, Blundell notes that Bio-Rad’s StarBright Dyes were developed to address common flow cytometry problems as well as provide researchers with more fluorochromes to select from. “StarBright Dyes are available excited by the 355 nm, 405 nm, 488 nm, and 561 nm lasers,” he says. “They are characterized by superior brightness, to increase researchers’ chances of detecting rare/low antigen density populations, and feature narrow excitation and emission profiles, to minimize spillover. StarBright Dyes are also easier to use than many older fluorochromes. Because they do not require special buffers or staining protocols, they are readily integrated into existing panels. And because they can be premixed for over 30 days, they enable the same cocktail to be used for staining multiple samples, which can save time and reduce error.”

Five tips for successful multiparameter flow cytometry

• Understand the biology of your chosen model system, including factors such as antigen density, co-expression, and experimental conditions
• Check the capabilities of your flow cytometer
• Consider making a list of commercial antibody conjugates to clarify the options available for panel design
• Be rigorous with experimental optimization, including both the cell preparation method and the staining protocol
• Think about how you will analyze your data, and remember to include appropriate controls

Streamlining experimental design

Designing a multiparameter flow cytometry experiment should be a methodical and data-driven process. Blundell recommends that you first establish which markers are required to detect the cell types of interest. “Factors to consider include the number of markers needed for definitive identification, the antigen density, whereabouts on (or within) the cell the markers are expressed, and whether some form of treatment is needed to stimulate marker expression,” he says.

At the same time, you will also want to think about the capabilities of your flow cytometer. “Specifically, if you are using a conventional flow cytometer, you will need to check which lasers and detection channels your instrument is fitted with since this will determine which fluorochromes you are able to use and the maximum number of parameters you can analyze in the same experiment,” says Guenther. For spectral flow cytometry, as long as the fluorochrome is excited by the laser, it will be compatible.

Next, you should assign fluorochromes to the different markers. “Building a list of available fluorochromes for your antibodies of interest can save time here,” advises Duncker. “Often, less commonly used markers will have fewer commercial fluorochrome options, so it can be prudent to assign fluorochromes to these markers first, before building the remainder of the panel.” Guenther also suggests strategically choosing fluorochromes to align with antigen abundance. “If you have an antigen on a cell that is in low abundance, it is best to pair it with a fluorochrome that is very bright, and vice versa,” he says.

After this, it is essential that you optimize your experimental conditions, including both the cell-preparation method and the staining protocol. This should include identifying a suitable cell concentration, implementing measures to avoid cell death or clumping, and determining whether sequential staining is necessary to prevent extracellular epitopes from being damaged by fixation and permeabilization.

Finally, you must decide how the data will be analyzed to get the most out of your hard-won results. “Choices here include whether to perform serial gating with two-color plots, or whether to use unsupervised clustering algorithms,” says Blundell. “If in doubt, manufacturers of flow cytometry instrumentation and reagents can often offer guidance and share links to relevant resources and publications.”