Flow cytometry depends on a combination of fluorophores and target-specific antibodies as the detection reagents. Many antibodies used in flow cytometry are directly conjugated to fluorescent dyes (aka fluorophores). However, many unlabeled primary antibodies are also routinely used in combination with labeled secondary antibodies. Read on to learn how to select flow cytometry dyes including quick tips for flow cytometry dye selection such as matching fluorescence profiles of fluorophores to the configuration of flow cytometers, the factors that can affect fluorophore performance in a multicolor panel, and helpful tools for simple dye selection.
Fluorophores are either natural or artificial molecules that can be excited by light at specific wavelengths and can subsequently emit a lower energy light with a longer wavelength. The excitation and emission profiles are distinctive features of each fluorophore. Therefore, fluorophore selection is a key step in flow cytometry.
The terms “fluorophore” and “fluorochrome” are often used interchangeably in the literature on flow cytometry. However, “fluorophore” generally refers to the individual molecules that fluorescence themselves, as described above. Whereas “fluorochrome” refers to the fluorescent dyes used in the biological staining before flow cytometry investigation.
It is essential to match the fluorescence profile of each fluorophore with the optical configuration of the flow cytometer. As a first step, it is important to know the configuration of the flow cytometer in use, such as the number and the types of lasers and filters it is equipped with. The lasers must emit light that is close to the maximum excitation wavelength of the fluorophore(s) that are selected. In addition, the filters must be suitable to detect the light that is nearest to the maximum emission wavelength of the fluorophore(s).
Over the past decade, the list of fluorophores or fluorescent dyes suitable for flow cytometry has grown significantly to help satisfy the need for simultaneous detection of multiple markers in a given cell population. This has led to the emergence of high-throughput or multicolor flow cytometry analysis that offers the advantages of:
The desire for multicolor flow cytometry experiments has posed a few challenges. Researchers must use several criteria for selecting appropriate dyes or fluorophores to avoid any erroneous results when designing their multicolor flow cytometry panels. Where a panel of fluorophores is used, it is best to distribute the fluorophores as widely as possible across the lasers and filters to reduce any possible interference in the analysis.
The fluorophore performance and its utility in a multicolor panel depend on the following factors:
The following few tips can be useful in selecting suitable fluorescent dyes or fluorophores in flow cytometry analysis.
We are proud to offer a constantly expanding selection of our unique ColorWheel® flow cytometry antibodies and dyes. The antibodies and dyes use proprietary oligo-based technology that allows for the mix and match of any antibody to any dye. These reagents offer enhanced simplicity when compared to secondary conjugated antibodies and labeling kits.
Any of the ColorWheel® dyes that are offered can be conjugated to any of the ColorWheel® flow cytometry antibodies with a quick and simple protocol in less than 30 minutes at ambient temperature. This makes these reagents the perfect tool for simple dye selection, especially when your experiments call for multicolor flow cytometry panels.
Learn more about how easy it is to prepare ColorWheel® flow cytometry antibodies and dyes in our 3-Step ColorWheel® Flow Cytometry Reagent Preparation protocol. Or, see how to incorporate ColorWheel® products into your flow cytometry experiment in our ColorWheel® Flow Cytometry protocol.