Multicolor Flow Cytometry: Tips and Tools for Building Flow Cytometry Panels

Flow cytometry is a powerful technique for characterizing and/or sorting heterogeneous suspended cell populations on the basis of their physical and fluorescence characteristics.  With the development of new fluorochromes that are suitable for flow cytometry, interest in multicolor flow cytometry has grown immensely. Read on to learn more about the challenges in multicolor flow cytometry, steps for how to build a multicolor flow cytometry panel, tandem dyes, and tools for multicolor assay design.

• What Is Multicolor Flow Cytometry?
• Challenges with Multicolor Flow Cytometry
• How to Build a Multicolor Flow Cytometry Panel
• Using Tandem Dyes

What Is Multicolor Flow Cytometry?

Multicolor flow cytometry is a rapidly evolving technology that uses multiple fluorescent markers to identify and characterize cellular subpopulations of interest.  It improves the efficiency of flow cytometry experiments by requiring fewer samples and smaller sample volumes. Increasing the sample throughput can allow one to study drug effectiveness towards different cell types and analyze various protein-protein interactions. Although smaller flow cytometry panels minimize the complexity of spillover and instrument compensation, more sophisticated and advanced flow cytometers have allowed more parameters to be studied simultaneously and more complicated questions to be answered.

Multicolor flow cytometry, also known as multiplex flow cytometry, has become an essential tool for studying the immune system in health and disease studies because it allows researchers to extract multiparametric data from smaller amounts of samples. However, the process may be complicated by substantial variations in the performance of different flow cytometers. Hence, one must systematically pair fluorophores in each multicolor panel to run on the flow cytometer of choice.  Data obtained in multicolor flow cytometry experiments also require careful analysis.

Challenges with Multicolor Flow Cytometry

Although multiplexing in flow cytometry offers several advantages over the use of only one primary-conjugated antibody per sample, multicolor flow cytometry does have its own challenges.

These challenges include:

• It requires detailed experimental design including fluorochrome selection and target considerations.
• Multicolor panel design requires a careful assemblage of fluorochromes with non-overlapping spectra.
  • This also includes the need for compensation and any “spillover” adjustments to obtain accurate, reliable, and reproducible data. 

How to Build a Multicolor Flow Cytometry Panel

In order to build a reliable multicolor flow cytometry panel and avoid issues with spectral overlap and loss of resolution, one should design a panel using a spillover spreading matrix (SSM) generated from the flow cytometer to be used. The SSM is used to ensure that fluorochromes with significant spectral overlap do not associate with the markers expressed on the same cell type or to assist in detecting markers that are present in extremely low levels. The SSM also allows one to ensure that dim colors are used for highly expressed markers and vice versa. 

Usually, researchers rely on their own experiences regarding the markers in question and the expected antigen density. Although manufacturers may provide the antibody affinities on their websites, it is a best practice to test and verify the antibody binding of each new lot that is purchased. Once the antibodies have been tested, the full multiplex flow cytometry panel should be tested on healthy cells.

Steps in Building a Multicolor Flow Cytometry Panel

Designing and building a multicolor flow cytometry panel is one of the most difficult processes when researching with multiplex flow cytometry assays. It involves the following steps:

• Carefully select the antigen targets.
  
• Confirm knowledge of the configuration of the instrument being used (both laser and filters).
  • The profile of the selected fluorochromes should match the optical configuration of the flow cytometer.
• Perform a literature review or use online spectra viewers to examine any spectral overlaps.
• Optimize fluorophores as widely as possible across lasers and filters to reduce any possible interference.
• Consider the Stokes shift for each fluorochrome used in the multicolor panel.
• Although fluorophores with non-overlapping emission spectra are ideal, flow cytometry compensation controls or control beads may be used to ensure that any detected light is ascribed to the correct fluorophore.
• Select and reserve the brightest fluorochrome for the weakest antibody and vice versa.
• Research the literature to explore cell populations expressing the particular targets of interest and the degree of expression of each of the targets.
• Check to see if any specific cell line or cell stimulation is required to generate a signal.
• Review the literature to investigate which subpopulations of cells co-express the same markers.
• Select the appropriate controls.
  • Unstained cells may be used as a negative control population.
• Optimize each antibody concentration to be used.
  • Titrate to the concentration that generates the largest spread with the least amount of background.
• Consider the photostability of each antibody-conjugated fluorophore.
• Decide which clone works best if multiple clones of monoclonal antibody are available.
• Stain cells with the full flow cytometry panel and evaluate. Make changes if necessary.
• Always include a cell viability dye in the panel to assess cell death during the course of the experiment.

Using Tandem Dyes

The use of tandem dyes is common in flow cytometry analysis. However, they are subject to degradation, either due to photosensitivity or long-term storage. This leads to decreased Fluorescence Resonance Energy Transfer (FRET). It also leads to an enhanced signal from the acceptor and donor fluorophores, which demands increased compensation and may generate unreliable data.

Some tandem dyes are also sensitive to fixation. It is also possible that some of these dyes may bind non-specifically to certain cell types, such as the PE-Cy5, PerCP-Cy5, and APC-Cy7 dyes that bind to monocytes and macrophages, in part due to their binding to the human high-affinity FC receptor CD64.