HomeFlow CytometryFlexibility in Multiplexing: ColorWheel® Antibodies and Dyes

Flexibility in Flow Cytometry Multiplexing with ColorWheel® Antibodies and Dyes

Flow cytometry is a technique that allows researchers to rapidly analyze individual cells within a heterogeneous population and is frequently utilized within immunology, immuno-oncology, neurobiology, and many more fields of research. The additional ability to evaluate multiple characteristics of these cells through assays utilizing various fluorochromes or dyes makes multiplexing in flow cytometry a very dynamic and powerful tool. However, designing and expanding multicolor assays come with a variety of challenges that researchers must overcome. Our ColorWheel® antibodies and dyes are specifically designed to address these challenges and bring simplicity and flexibility to multiplex flow cytometry assays without compromising on performance.

What Is Multiplexing in Flow Cytometry?

Multiplexing or multicolor flow cytometry refers to the ability to recognize different cell surface and intracellular antigens with multiple antibodies labeled with fluorescent markers within a single well of a microtiter plate. Cells are hydrodynamically focused into a single stream and passed through a laser beam, allowing for multiparameter data collection of each cell. By adding a unique fluorochrome or dye to each antibody, one can distinguish and characterize cellular subpopulations of interest by the color(s) emitted when excited by the cytometer’s laser beam(s)1 .

Factors to Consider When Building a Flow Cytometry Multiplex Panel

The ability to analyze multiple antigens simultaneously opens the door to increasingly complex experiments, and often routine experiments using the same antigens, commonly referred to as panels. When building a multiplex panel in flow cytometry, there are numerous factors that researchers must consider:

ColorWheel<sup>®</sup> antibodies and dyes help solve the flow cytometry multiplexing puzzle by fitting together the different pieces or challenges of multiplexing in flow cytometry including instrument configuration, antigen expression/dye brightness, and antibody-dye combination availability.
  • Instrument configuration: The wavelength of lasers available on the instrument must be compatible with the fluorochrome or dye labeled to the antibody, with detection of these fluorescent markers further controlled by filters.
  • Antigen expression, cell population abundance, and dye brightness: In general, a good strategy is pairing high-intensity dyes with rare populations of cells or antigens with patterns of low expression2 . There are exceptions to this rule, so a deep understanding of antigens, cell populations, and dyes within the experiment is critical for valid results.
  • Antibody-dye combination availability: Find an antibody against the antigen of interest labeled with a dye that works with your instrument from a vendor that has validated the antibody’s use in flow cytometry. If this combination is not commercially available, indirect staining using a secondary antibody can be used. However, this may introduce species cross-reactivity, high background noise, and ultimately more complexity – specifically in multiplex applications.

The balance of these aspects to build a new multiplexed experiment can be complicated and take quite a bit of time and effort to achieve. Expanding existing multicolor panels to address new questions adds another layer of complexity to this already daunting task. Researchers must find a flow cytometry-validated antibody against the antigen of interest with a dye appropriate for that antigen’s expression level, not already in use by the instrument’s laser configuration, and without significant spectral overlap with existing antibody-dye pairs in the panel.

Explore tips on how to build a multicolor flow cytometry panel in our guide to multicolor flow cytometry.

Multiplexing with ColorWheel® Antibodies and Dyes

When solving the puzzle of multiplexing in flow cytometry, the simplicity, and flexibility that comes with ColorWheel® antibodies and dyes remove experimental constraints to unlock more freedom in multiplex assay design. Using proprietary technology, any ColorWheel® antibody can be paired with any ColorWheel® dye. All ColorWheel® antibodies and dyes are validated for use in flow cytometry and are optimized to work at convenient 1:1 (volume/volume) ratios for easy assembly of antibody-dye pairs. With just three steps and five minutes of hands-on time, users can create optimal panels of antibody-dye pairs for their instrument with antigens of interest, using our Simple 3-Step ColorWheel® Flow Cytometry Reagent Preparation Protocol.

Relative cell counts of ColorWheel® CD44, CD4, CD45RA, and CD3 antibodies paired with ColorWheel® AF450, AF488, PE, and APC dyes respectively. These were run simultaneously in a flow cytometry multiplex format.

Figure 1.ColorWheel® CD44, CD4, CD45RA, and CD3 antibodies paired with ColorWheel® AF450, AF488, PE, and APC dyes respectively were run simultaneously in a multiplex format.

Consider the following scenarios:

Expanding an Existing Multiplex Assay by Adding a New Target

Problem: The new target is only commercially available conjugated to one dye, displacing the antibody already conjugated to that dye in your existing multicolor assay.

ColorWheel® Solution: Instead of forcing users to decide which target to analyze, the mix-and-match ability of ColorWheel® antibodies and dyes provides freedom in target choice by allowing a simple exchange between the antibodies already in the assay with any unused instrument-compatible ColorWheel® dye. By building panels of ColorWheel® antibodies and dyes, multiplex flow cytometry screening assays become “futureproof”, ensuring that existing dyes can always be rearranged with new or existing antibodies in the multicolor panel.

Building a New Multiplex Assay with Antigens of Varying Expression Levels

Problem: Matching antibodies against antigens of varying expression levels with dyes of suitable brightness consumes a lot of time and effort.

ColorWheel® Solution: The user-friendly ColorWheel® portfolio allows you to order the targets you want with the dyes that fit your instrument. Then, with the flexibility of ColorWheel® antibodies and dyes, simply mix and match your target and dyes at the bench as needed, freeing you from compatibility restraints. Further, with the 5+ years enhanced stability of ColorWheel® products, instrument-compatible dyes can always be on hand.

See how to use ColorWheel® products in our ColorWheel® Flow Cytometry Protocol. Or, learn more about finding the right fluorochromes for your panels with our guide on selecting the right fluorochromes.

Flexibility Without Compromising Quality: Comparison to Directly Conjugated Antibodies

ColorWheel® antibodies and dyes offer flexibility in your reagents without trading off the quality of your data. Compared to traditional directly conjugated primary antibodies, ColorWheel® antibodies show equivalent performance when run in multiplex assays (Figure 2).

Histograms showing relative cell counts of ColorWheel® antibodies paired with ColorWheel® dyes compared to traditional directly conjugated primary antibodies against the same targets, AF450, AF488, PE, and APC. Run simultaneously in flow cytometry multiplex format.

Figure 2.ColorWheel® antibodies paired with ColorWheel® dyes compared to traditional directly conjugated primary antibodies against the same targets. These were run simultaneously in a multiplex format.

Stay Connected to Discover More

Stay connected with us by scanning the convenient QR code printed on each ColorWheel® product label to take you to our ColorWheel® digital hub. Here you will find the newest additions to the ColorWheel® portfolio, as well as information and resources on the technology, applications, and more.

Our ColorWheel® antibodies are designed and tested with your needs in mind and are backed by our Antibody Guarantee. You can also experience them for yourself by requesting more information about our trial offer.

Related ColorWheel® Products

You need both a ColorWheel® antibody and a ColorWheel® dye for your flow cytometry analysis. The antibody and dye are sold separately.



Maciorowski Z, Chattopadhyay PK, Jain P. 2017. Basic Multicolor Flow Cytometry. Current Protocols in Immunology. 117(1):
Maecker HT, Frey T, Nomura LE, Trotter J. 2004. Selecting fluorochrome conjugates for maximum sensitivity. Cytometry. 62A(2):169-173.
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