A Guide to Spectral Flow Cytometry Fluorophore Selection

Herein we provide guidance on how to select fluorophores and other reagents for spectral flow cytometry.

Design and build spectral panel  Spectral flow cytometry fundamentals

Advances in flow cytometry instrumentation coupled to a growing number of fluorescent labels and readouts have expanded applications and capabilities beyond conventional flow cytometry. Now with spectral flow cytometry analysis, researchers and scientists can investigate an increasing number of molecules of interest. Importantly, larger polychromatic panels still require careful selection of available fluorophores, fluorescent reagents, and proteins. Here we provide spectral flow cytometry users a guide to selection of available fluorophores, assays and reagents that fit the needs of multiplexed spectral flow cytometry approaches in cell biology, immunology, cancer biology, microbiology, and plant biology.

As the flow cytometry research community strives to increase panel complexity, different enabling technologies are becoming available to meet their scientific needs. Researchers are starting to turn to spectral flow cytometry analyzers, such as the Cytek Aurora spectral flow cytometer provided by Cytek Biosciences or the SA3800 provided by Sony Biotechnology . Spectral flow cytometers exploit the inherent emission pattern of each fluorescent molecule to generate a unique spectral signature (comparative examples in Figures 1–3). However, each instrument has different laser configurations and optical sensitivity. It is critical to understand these differences when designing multicolor panels and choosing the appropriate fluorochrome combinations. In any case, individual fluorescent reference controls are needed, similar to that of single-color controls in conventional flow cytometry, in order to deconvolute or unmix the spectral signatures in polychromatic panels. Therefore, spectral flow cytometry analysis relies on the discrimination of unique spectral signatures rather than specific emission channels for detection, enabling the compatibility and distinction of many fluorescent combinations that were previously difficult or impossible to separate, as shown by PerCP and PerCP-eFluor 710 (Figure 1).

Note: Each spectral signature (Figures 1-3) generated displays the distribution of events as a function of intensity across the whole spectrum, for each laser. Interpreting the color scheme for each channel is similar to that of a heat map or density plot. Red represents the intensity at which most events had fluorescence, while yellow, green and blue represent decreasing numbers of events. Each laser’s set of detectors can have additional emission captured enabling the distinction between fluorophores, as indicated by the red boxes. However, based on this information dyes that emit at similar wavelengths can introduce spread into the other. Refer to Tools to achieve effective panel design for information on the amount of spread introduced among fluorophores.

A

B

Figure 1. Comparison of a large protein molecule and its tandem. (A). Overlay of PerCP and PerCP-eFluor 710 excitation and emission profiles, demonstrating their significant overlap, taken from Fluorescence SpectraViewer. (B). PerCP positive cells (top graph) compared to PerCP-eFluor 710 positive cells (bottom graph) analyzed on a 3-laser spectral flow cytometer* system. Although emission values are very similar, the unique patterns in the far-red channels allow for the two molecules to be discriminated.

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Various classes of fluorescent probes and reagents can be multiplexed more readily in spectral flow cytometry experiments. For instance, in larger immunophenotyping panels when available labeling reagents become limited, the Invitrogen Alexa Fluor 647 dye can now be used in combination with larger fluorescent proteins such as with APC (Figure 2). When expanding panels to identify sub lineage cell types and associated markers, the violet laser-excitable Super Bright antibody conjugates can be taken advantage of in combination with traditional dyes like eFluor 450 and Pacific Orange. Flow cytometry antibody panels are the basis for immunophenotyping experiments, but inclusion of functional reagents such as CellTrace can be challenging in larger panels on conventional instrumentation. Spectral flow cytometers can allow for simpler integration of functional reagents by utilizing their unique spectral signatures. Our Invitrogen portfolio of fluorescent reagents and assays are the perfect complement to the burgeoning field of spectral cytometry analysis that can enable cell biologists, immunologists, and cancer biologists the ability to uncover deeper biological insights.

Figure 2. Example of two compatible fluorochromes. Allophycocyanin (APC) (top graph) and Alexa Fluor 647 (bottom graph), are now compatible when analyzed on a 3-laser spectral flow cytometer*. Although emission profiles are similar, their unique patterns highlighted in the violet and blue channels allow for the molecules to be discriminated.

Invitrogen fluorescent probes are capable of elucidating biological mechanisms such as cell cycle stages, important cell signaling pathways, cell death through apoptosis, RNA detection, etc. Combining these probes to antibody staining requires careful calibration and the appropriate biological controls. As cellular autofluorescence can interfere with fluorescence detection in some protocols, spectral flow cytometry is capable of identifying and removing autofluorescence during analysis (see Figure 3 for an Invitrogen PrimeFlow RNA example). During spectral unmixing autofluorescence can be removed in order to better resolve and identify the true signal from the fluorescent probe of interest. This capability further enables combinatorial approaches of antibody labeling with fluorescent-based functional probes to elucidate the biological significance during spectral flow cytometry experiments.

Figure 3. Example of autofluorescence extraction. PrimeFlow RNA detection was used to label mRNA in human U937 cells. Cells were treated with PrimeFlow RNA detection kit and were either unstained or stained before detection on a 3-laser spectral flow analyzer* (Panel A). Unstained cells were mixed with stained cells and analyzed before and after autofluorescence removal (Panel B).

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Like traditional flow cytometry, spectral flow cytometry enables live cellular analysis, which is critical when investigating numerous immunological areas. For instance, when interrogating the tumor micro-environment, flow cytometric assays are useful approaches to cytokine profiling, assessing tumor specific antigens, immune checkpoint discovery, CAR T cell therapy, among others. Careful panel design for spectral flow cytometry analysis requires an understanding of an instrument’s capabilities, cell lineage sub populations, their expected antigen density, and the available antibody-conjugates and their properties. The information provided in the following tables is intended to guide researchers in their selection of fluorochromes for polychromatic panels when using the Cytek Aurora 3-laser system.

Table 1. Spread matrix of 20 Invitrogen fluorophores that can be used simultaneously in spectral flow cytometry. A fluorescent dye matrix that demonstrates the level of spread among dyes. Fluorophores of each row impact the spread of the fluorophore in the column. Although all dyes in the matrix can be used together, the darker red shading means one fluorophore has increased spread into the other and needs closer attention when designing panels and interpreting data.

Click on each cell to see comparisons of spectral signatures and the percent reduction of the dye's cross-staining index. The comparison of spectral signatures is a good indicator of the impact that one fluorophore can have on the other, while the percent reduction of the dye's cross-staining index is intended to be used as a quantitative measurement of resolution. By understanding the influence of one fluorophore's spread (row) into the resolution of the other (column), the selection of appropriate fluorophores that can be used in combination is much simpler. In this matrix all fluorophores were controlled for the same antigen (CD4) and is intended to be a valuable point of reference in panel design.

Table 2. Spread matrix of 24 fluorophores that can be used simultaneously in spectral flow cytometry. When expanding the panel with additional fluorophores refer to the fluorescent dye matrix below, which demonstrates the level of spread among dyes. Fluorophores of each row impact the spread of the fluorophore in the column. Although all dyes in the matrix can be used together, the darker red shading means one fluorophore has increased spread into the other and needs closer attention when designing panels and interpreting data.

Table 3. Invitrogen reagents that are not recommended to be used together in spectral flow cytometry. All fluorophores in the spread matrices above are usable together. However, based on the needs of the experiment and the available antibody conjugates, adjustments might need to be made. Refer to the table below as a guide when customizing your panel. Each cell contains a list of fluorochromes and viability dyes that are not recommended to be used together.