Mastering Fluorochrome Selection for Flow Cytometry

When designing a multicolor flow cytometry panel, researchers have almost limitless choice in deciding which fluorochromes to use. However, not all fluorochromes are created equal, and it is important to understand how different fluorochrome properties can impact the quality of flow cytometry results. Whether you’re performing traditional flow or using a spectral instrument with advanced imaging capabilities, the following strategies can help simplify panel design, reduce spillover, and maximize the resolution of your target population.

Plan for success

Before embarking on panel design, you should define your experimental hypothesis, clearly outlining the type of biological information you are trying to capture. This includes deciding which cell populations you wish to interrogate, establishing which markers are required for their identification, and confirming where, when, and how those markers are expressed. Determining whether you will need to stain for both cell surface and intracellular markers is a key consideration. If such detection is required, staining for extracellular targets first—prior to fixation, permeabilization, and intracellular staining—is generally advised to preserve cell surface epitopes.

You should also make sure to understand the capabilities of your flow cytometer, since instrument specifications will determine how many markers can be measured simultaneously and which fluorochromes it is possible to detect. Factors to consider include the laser wavelengths used for excitation and whether the platform is built to support traditional or spectral flow cytometry. While traditional flow cytometers limit the choice of fluorochromes to those that are compatible with the system’s optical filters, spectral flow cytometers allow researchers to use any fluorochrome that is excited by the instrument’s lasers, increasing flexibility for panel design.

Consider marker expression levels

Although some cell populations can be identified using just one or two markers, it is more common for multiple markers to be combined. To streamline the process of assigning fluorochromes to different markers, it is suggested that you classify each marker as a primary, secondary, or tertiary antigen based on expression levels, as detailed below. This will help to ensure that the most difficult to detect antigens—which usually have the fewest commercially available antibody conjugates—are addressed first, making it easier to subsequently identify other antigen-fluorochrome pairings.

• Primary antigens are usually well-characterized and typically exhibit expression that is either on or off; they are commonly used for defining cell lineages and include CD3, CD4, and CD19.
• Secondary antigens also tend to be well-characterized, but are more often expressed over a continuum; they enable deeper interrogation of cellular subtypes and include CD27, CD28, and CD45RA.
• Tertiary antigens are expressed at low density and are critical for cellular identification; examples include CD25, STAT5, and FoxP3, in addition to uncharacterized antigens.

Understand fluorochrome performance characteristics

Fluorochromes have several inherent properties that distinguish them from one another. These include characteristic excitation and emission spectra, which respectively have an excitation peak (λex) and an emission peak (λem), and a Stokes shift, which represents the difference between λex and λem. Fluorochrome brightness is another important feature, defined by the molar extinction coefficient (ε) and the quantum yield (Φ), which respectively indicate how strongly the fluorochrome absorbs light at a specific wavelength and how efficiently that light is emitted.

Fluorescence spillover, which defines the spectral overlap between the emission profiles of two fluorochromes, and fluorescence resolution, which defines the degree of separation between the negative and positive populations, are two of the most critical factors when it comes to choosing fluorochromes for flow cytometry. Products such as BD Horizon™ Real Fluorochromes are specifically designed to minimize fluorescence spillover and optimize panel resolution, and are easily incorporated into flow cytometry panels using BD Biosciences’ Fluorochrome Performance Guide, which organizes and ranks fluorochromes based on these characteristics.

For researchers running experiments on the BD FACSDiscover™ A8 Cell Analyzer or BD FACSDiscover™ S8 Cell Sorter, BD Biosciences’ Fluorochrome Selection Guide aids the identification of optimal reagents for applications with and without imaging.

Tips for fluorochrome selection

When assigning fluorochromes to markers, there are several strategies that can be applied to ensure you resolve markers at all expression levels and minimize spectral overlap. First, aim to pair bright fluorochromes with low expressing antigens, and vice versa, using tools such as the Fluorochrome Performance Guide and an online spectra viewer to evaluate factors including cross laser excitation and the potential for spillover. Second, try to spread fluorochromes across as many lasers and detectors as possible to minimize the risk of unwanted background signal; if the number of lasers becomes restrictive, you may wish to consider using tandem dyes to increase the number of fluorochromes excited by each laser. Other factors to consider include fluorochrome size, especially when performing intracellular staining, and fluorochrome stability, which is particularly critical when planning longitudinal studies.

Conclusion

While the process of selecting fluorochromes for flow cytometry can sometimes seem overwhelming, the development of advanced fluorescent reagents and easy-to-use panel design tools is helping to drive experimental success.