HomeFlow CytometryFlow Cytometry Dye Selection Tips

Flow Cytometry Dye Selection Tips

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.

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What Are Fluorophores?

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.  

Fluorophore vs Fluorochrome

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.

Matching Fluorescence Profiles to Configuration of Flow Cytometers

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). 

Factors Affecting Fluorophore Performance in Multicolor Panels

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:

  • Generating a detailed analysis of the cells in a mixed cell population
  • Allowing researchers to study drug effectiveness towards different cell types
  • Enabling the analysis of protein-protein interactions 

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:

  • Excitation and emission profiles
  • Relative brightness
  • Quantum yield
  • Emission overlaps
  • Stability of antibody-fluorophore conjugate
  • Reproducibility of antibody conjugation

Tips for Selecting Appropriate Fluorescent Dyes

The following few tips can be useful in selecting suitable fluorescent dyes or fluorophores in flow cytometry analysis. 

  • Ensure the fluorophore profile matches the optical configuration of the flow cytometer.
  • Always take into account the excitation and emission maximums as well as the Stokes shift in designing your flow cytometry experiments when using a panel of antibodies.  
  • Choose the brightest set of fluorophores for your flow cytometer instrument configuration. 
  • Choose fluorophores to help minimize the spectral overlap. 
  • Reserve the brightest fluorophores for the weaker antibodies and vice versa. 
  • Avoid spillover from the bright cell populations into detectors that require high sensitivity for those populations. 
  • Consider the photostability of each fluorophore as well as its stability after fixation depending on the experimental needs. 
  • Although using fluorophores with non-overlapping emission spectra is ideal, compensation controls or control beads may be used to ensure that any detected light is ascribed to the correct fluorophore when multiplexing.  
  • Confirm that there is limited or no sensitivity to the other reagents used, such as lysing agents, fixatives, etc. 

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