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.
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.
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:
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.
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:
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.
We offer a wide selection of mix-and-match ColorWheel® flow cytometry antibodies and dyes that are designed to provide greater flexibility in multicolor assay design and expansion over traditional pre-conjugated primary antibodies. ColorWheel® antibodies and dyes were designed to help remove the experimental constraints and challenges involved in building a multicolor flow cytometry assay.
Any ColorWheel® antibody can be easily paired with any ColorWheel® dye using proprietary oligo-based technology. These reagents are optimized to work at a convenient 1:1 (volume: volume) ratio for the simple assembly of antibody-dye pairs. Preparing the reagents takes three easy steps and five minutes of hands-on time to help create optimal multicolor panels of antibody-dye pairs with the respective antigens of interest.
Learn more about how ColorWheel® flow cytometry antibodies and dyes can be used to enhance multicolor flow cytometry experiments in our Flexibility in Multiplexing article. Or, see how to incorporate ColorWheel® products into your flow cytometry experiment in our ColorWheel® Flow Cytometry protocol.