How to Build Complex Multicolor Flow Cytometry Panels

Flow cytometry is a powerful technique that allows the identification and characterization of cell populations in a sample via their protein expression. Ongoing advances in instrumentation, alongside the development of novel dyes, have enabled the use of bigger, more complex panels.

Of course, this is great in the sense that you can get more information and answer more questions from a single sample than ever before; however, building these panels can be complicated and overwhelming, especially if you’re new to the technique.

Thankfully, this is where we come in! In this blog, we’ve compiled our top tips and steps to follow when designing multicolor flow cytometry panels.

Need urgent advice? Skip straight to the “Quick Tips” section for a summary.

Action Plan

First, you will need to do some planning. While it may be tempting to throw in as many markers as possible, this can complicate downstream analysis, so it’s better to be intentional with your decisions.

Keep in mind the questions that you intend to answer when planning your experiment and establish how you will use flow to obtain those answers.

Once you know your target cell populations, you can determine which markers you need to identify them. It’s best to use well-established cell-specific markers for reliable identification, so searching the literature for previously published gating strategies is a good way to decide which markers to use. Alternatively, using online tools, such as Bio-Rad’s interactive Immune Cell Markers Selection Tool, can be a quicker method to choose appropriate markers.

Figure 1 shows how a carefully designed gating strategy can identify multiple cell subpopulations with only five colors. Lymphocytes, monocytes, and granulocytes have distinct cell characteristics. Therefore, it is possible to separate them based solely on their forward scatter (FSC) and side scatter (SSC) properties, which give an indication of the cell size and granularity, respectively (Figure 1A).

Within the monocyte gate, CD14 and CD16 can be used to identify classical, non-classical, and intermediate subsets (Figure 1B), while within the lymphocyte gate, helper and cytotoxic lymphocytes can be identified using CD3, and CD4 and CD8, respectively (Figure 1C and D).

Fig. 1. A simple gating strategy to identify multiple populations using only five colors.    

Color Coordinated

As you add more markers and thus more colors to your panel, the complexity rises as you need to avoid spectral overlap. In a two-color panel, it’s simple to choose colors at opposite ends of the visible light spectrum, but it becomes more difficult to find nonoverlapping spectra when you are working with more dyes.

The excitation and emission spectra of each dye are typically found on the product datasheet, so you can note the profiles of each of your dyes individually and plan your panels this way. However, an easier way to avoid spectral overlap is to use an online spectraviewer tool, which lets you visualize all the spectra of your dyes at once.

You can see an example of Bio-Rad’s Spectraviewer in action in Figure 2. Panel A shows the excitation and emission spectra for StarBright Violet (SBV) 515 Dye. In panel B, you can see that adding Brilliant Violet 510 to the panel would result in a significant overlap of the spectra, therefore, this should be avoided. Instead, SBV710 (panel C) can be used, as it has a distinct emission spectrum from SBV515 despite being excited by the same 405 nm laser.

You can increase the number of dyes in your panel by using multiple lasers for excitation. It’s important to understand the configuration of your cytometer to know which lasers, detectors, and filters are available to you.

Spectraviewers often allow you to input your instrument’s configuration to facilitate the identification of fluorescent dyes compatible with your laser combinations. Panel D demonstrates that PE is compatible with this panel, as it is excited by the yellow 561 nm laser, and the emission spectrum does not significantly overlap with those of SBV515 and SBV710.

Choosing dyes with narrow excitation and emission spectra, such as Bio-Rad’s StarBright Dyes, allows you to build bigger panels while still avoiding spectral overlap.

Fig. 2. Bio-Rad’s Fluorescence Spectraviewer can help you avoid spectral overlap in your panel. The dotted curve represents the excitation spectra, with the peak representing the best wavelength to maximally excite the fluorescent dye. The filled curve represents the emission spectra.

While it is always best to reduce overlap as much as possible, the extent to which dyes can be separated differs significantly depending on whether you are using conventional or spectral flow cytometry, so it’s important to know this information in advance of building your panel.

In conventional flow cytometry, a single detector captures the peak emission of a dye, so dyes with similar emission peaks cannot be used simultaneously.

In spectral flow cytometry, however, the entire spectrum is captured using all the detectors, generating a unique spectral “fingerprint” for each dye. This greatly increases the ability to distinguish overlapping spectra, enabling the use of more dyes in a panel. For example, take a look at Bio-Rad’s 43-color human immunophenotyping panel!

Putting It All Together

So, you’ve determined which markers you want to detect and chosen the dyes that work best with your cytometer. How should you put these together?

There are several best practices that you should follow when building your panels. For example, you should reserve bright fluorescent dyes for targets with low antigen density, and dimmer dyes for targets that are expressed at high levels. You can find the relative brightness of commonly used fluorophores here.

As mentioned, spectral overlap should be avoided. However, this is not always possible. Therefore, dyes with significant overlap should be placed on markers that are not coexpressed in the same cell. This way, fluorescent spread will not mask a double-positive population.

To simplify this process, our online Multicolor Panel Builder Tool allows you to select your instrument configuration and the markers you are interested in, and it will automatically build a suitable panel, choosing dyes that work with your cytometer and pairing them with the most appropriate markers.   

In addition, all antibodies should be titrated to determine the optimal concentration prior to use, and all appropriate controls should be included to ensure confidence in the results.

If you’re a true beginner to the technique and are feeling uneasy about jumping in at the deep end, it may help to mimic the panels that you’ve seen in papers or tech notes that have the same target populations as you. There are also a variety of smaller, premade panels that can be built upon to create your ideal panel, without having to start from scratch.

Quick Tips

1. Plan your experiment carefully.
2. Determine your target markers using publications or marker selection tools.
3. Understand your cytometer’s configuration.
4. Decide which dyes to use based on minimizing spectral overlap — use a spectraviewer for ease.
5. Pair each marker with the appropriate dye, e.g., markers with low antigen density with bright dyes and vice versa — an online panel builder can help!
6. Titrate antibodies prior to use and utilize applicable controls.
7. Use suitable panels from publications, tech notes, or premade panels as a jumping-off point.

By following these simple tips, you should be a master panel builder in no time!