Building Successful and Complex Panels in Flow Cytometry

Flow cytometry panel building can be daunting, whether you’re working with a 4-color panel or a complex 30-color setup. This article explores common challenges faced by flow cytometry users, such as fluorochrome selection, the utility of tandem dyes and managing spectral overlap, to help optimize your panel design for more reliable results.

How do I choose which markers to include in my panel?

To select markers effectively, start by defining your research question and identifying the specific cell populations you need to analyze. Once identified, choose key markers that best define these cells and establish a hierarchical gating strategy to refine their selection.

You can also look at publication reviews in respected journals to get a sense of which markers have historically been used to identify your cell population. If you are phenotyping a new population, it may be best to include as many general markers as possible by using a screening tool or reagent set that allows you to assess hundreds of markers at once.

How does my instrument affect fluorochrome selection?

Your instrument’s optical configuration determines which fluorochromes are compatible. The excitation sources (the lasers) should match the fluorochrome’s maximal excitation range for efficient signal generation. For conventional flow cytometers, each laser must have corresponding filters to capture fluorochrome emissions.

Spectral flow cytometers are more flexible in this regard, but careful planning is still required to choose fluorochromes that have unique enough spectral signatures to be unmixed or separated.

What should I consider when choosing fluorochrome and antibody combinations?

When designing a flow cytometry panel, consider antigen abundance by using bright fluorochromes like PE and APC for dim markers to enhance signal detection. Conversely, highly expressed markers can be assigned dimmer fluorochromes. You may also want to put fluorochromes that are known to exhibit significant spillover on different cell populations to help avoid the issue altogether.

Be mindful of fluorochrome stability, as some dyes can undergo photobleaching or signal loss after fixation and permeabilization. In complex multi-color experiments, ensure proper fluorochrome separation and include appropriate controls for compensation or unmixing. Additionally, account for autofluorescence from cells or tissue, which can impact signal clarity and data interpretation.

Are tandem dyes a good option for expanding panels?

Tandem dyes are a great way to expand flow cytometry panels. In fact, tandem dyes are what allowed panels to first expand from 4–6 to 8–10 colors. They allow you to utilize multiple fluorochromes as the donor dye is excited by the same laser, while the acceptor dye emits at a longer wavelength due to the Stokes shift.

As with any dye, there are potential challenges like spectral overlap with neighboring dyes, which requires careful compensation. Tandems also can exhibit spillover into the channel of the donor dye (i.e., PE/Cyanine7 spilling back into PE). Some tandem dyes can also experience stability issues when exposed to light or fixative. In general, it can help to use a gentler solution with a lower percentage of fixative when working with tandem dyes.

What are some common troubleshooting issues when optimizing a panel?

Flow cytometry panel optimization depends on several key factors. Spectral overlap between fluorochromes can cause signal interference, requiring compensation or spectral unmixing to correct for spillover. Fluorochrome brightness and proper antibody dilution also impact signal detection.

At times, fluorochrome and antibody combinations that seem optimal on paper do not provide a strong signal in the assay. If you have antibodies that you know perform well in a panel, start with those and build outward by gradually introducing new ones. Add each antibody one by one to see how the new colors influence the overall spillover and compensation. Alternatively, test the antibodies whose performance you are unsure of first before committing to buying an entire panel of antibodies.

Regarding antibodies, it should be noted they can contribute to non-specific binding via the Fc portion of the structure, while some fluorochromes like PE/Cyanine5 can bind indiscriminately to myeloid cell types like monocytes. Both phenomena can potentially obscure true populations, and each benefit from the use of isotype controls and blocking solutions to help remove background noise.

From an instrument perspective, optimized settings ensure reliable data acquisition, and a well-defined gating strategy is essential for accurate population identification. Additionally, panel complexity must align with instrument limitations to avoid detection issues.

Lastly, variations in staining protocols can affect reproducibility and data quality, making consistency crucial in experimental design. If you are new to flow cytometry, you may want to try your hand at smaller panels and gradually build up the number of parameters instead of diving into a 20+ color panel.

What controls should I establish?

In flow cytometry, controls are essential for accurate data analysis. Unstained controls help establish baseline autofluorescence of cells. Single stain controls contain cells stained with only one fluorochrome to set compensation for spectral overlap. Fluorescence Minus One (FMO) controls include all fluorochromes except one to define gating boundaries for weakly expressed markers.

Positive and negative biological controls confirm antibody specificity and signal detection by using known expressing and non-expressing cell populations. This is particularly important if the sample only expresses a marker during transitory or activated states, i.e., IL-17 in Th17 cells. Viability dyes distinguish live from dead cells. For instrument controls, consider using compensation beads and calibration particles to check that your instrument is performing consistently.

Conclusion

Ultimately, we recommend that you talk to the right experts to set your panel up for success. Check with your advisors, lab mates and literature to find out how to best plan out your assays. Talk with reagent providers to learn about your fluorochrome and antibody choices. Speak with your flow core manager and/or instrument provider to learn what fluorochromes are best suited for your experiment. Once you are fully prepared, you will be ready to successfully build and run your optimized panel.