Cytometry and Antibody Technology

Spectral Experiment Design

Spectral Flow Cytometry

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Setting up a large panel on the Cytek Aurora or ThermoFisher Bigfoot can take some time. Unfortunately it’s impossible to predict the exact amount of time it will take, but a month is a very rough estimate. Keep in mind that there are many factors that could increase or decrease the optimization time, including panel size and user experience level. A general workflow for a typical spectral flow cytometry panel is outlined below:

  1. Design a panel using the cytometer-specific tools
    • Remember that panel design is theoretical and therefore it needs to be tested on actual cells
    • The harder the panel, the more time it can take to troubleshoot the panel
    • Consider running an unstained sample first to assess the autofluorescence signature. Depending on the level of autofluroescence in the sample it may be useful to consider autofluorescence in panel design.
  2. Titrate antibodies
    • Antibodies must be titrated using the full staining protocol that will be used for the final experiment.
    • Be sure to properly titrate fixable viability dyes. More information can be found in this blog post.
  3. Optimize Unmixing
    • The spectral unmixing resource offers tips for setting up and checking unmixing. It is critical to have correct unmixing before moving on to a panel check
  4. Check panel quality
    • It is important to assess the quality of the panel for every tissue used in the experiment. By assessing the spreading error within of the panel you can determine if all populations and markers of interest can be accurately separated from their respective negatives.
    • The panel design tutorial resource contains references for performing a panel quality check.
  5. Run experiment
    • Unmixing accuracy should be checked every single time – do not proceed to analyzing data until it is confirmed that no unmixing errors are present.


IMPORTANT NOTE FOR NEW SPECTRAL USERS: It is typically not recommended that users new to the Aurora or Bigfoot run their entire large panel as their very first experiment. It is fairly common for users to feel a bit overwhelmed by the large number of tubes, number of controls, new equipment, and new software. The best way to maximize your chance of success is to keep your first experiment small. For example, let’s say you plan on running a 20-marker panel. You should plan the panel for all 20 marker and you can start out with titrating all 20 markers if you wish. But once you move on to staining multiple markers in the same tube you should only choose 10 markers. This keeps the experiment to a manageable size and will allow you to work through the unmixing and panel check with a much higher rate of success. If the 10-color experiment is unsuccessful for any reason, continue to run practice experiments using only those 10 markers until you can produce excellent data. Once the 10-marker experiment can be successfully completed, you can expand your panel to the maximum number of markers. Depending on your level of experience, you may choose to jump from 10 markers to 20 markers, or you may find it more comfortable to move to 15 markers and then to 20 markers.


Controls on the spectral cytometers are comparable to controls on traditional cytometers. Single stain controls are necessary to calculate compensation. As discussed in the Aurora Training Course, it’s recommended to run one experiment with a double set of controls (both single stained cells and single stained compensation beads) in order to determine which combination of controls will produce the most optimal unmixing results. Any additional controls, such as FMO controls, are recommended and used in the same way as a traditional flow cytometry experiment.

Additional Considerations

Once a panel is properly set up on the Aurora, you must consider the design of the experiment. FMO controls are important in the same capacity as traditional flow cytometry. If you are repeating the same experiment and ultimately combining the data into the same final figure, you should also consider which instrument you choose. This is especially important given that the CAT Facility has multiple Auroras. The video below provides further details, but in short it’s easiest to always use the exact same Aurora.