Fluorescence Minus One (FMO) Controls

Author: Mike Blundell | Reviewer: Chloe Fenton

Fluorescence minus one (FMO) controls are important when building multicolor flow cytometry panels as they will help you determine where your gates should be set. This is particularly important when identifying a positive from a negative population and when the expression levels are low. When acquiring data, there is fluorescence spread, especially with brighter fluorophores, which is particularly noticeable after compensation, and cross-laser excitation. Careful experimental design and avoiding channels that have a large amount of spreading will help reduce this influence, but FMO controls are still important.

FMO controls are the experimental cells stained with all the fluorophores minus one fluorophore.

FMO Examples

An example of an FMO matrix is shown below in Table 4. Figure 22 shows how the fluorescence spread from other channels can affect the data. Using an FMO ensures you can position your gates accordingly, taking into account the fluorescence spread.

FMO controls should be done for all the fluorophores in your panel when starting a new multicolor experiment. This will then allow you to assess the spread of all the fluorophores into your missing channel and set your gates accordingly.

Table 4. FMO Matrix

Fig. 22. FMO controls to determine fluorescence spread. Dot plots of multicolor flow cytometry showing the fluorescence spread into the PE-Cy5 channel highlighted by the FMO control compared to an unstained control. The black dotted line represents the FMO gating boundary compared to the unstained boundary in red.

What Should You Do After Running FMO Controls?

• Define your gating strategy: use your FMO controls to correctly position gates and distinguish true positive populations
• Optimize your antibody panel: review fluorophore combinations to minimize spread and improve resolution
• Include additional controls: combine FMOs with compensation controls and biological controls for accurate interpretation
• Validate your experiment: confirm reproducibility across samples and conditions

Continue your workflow:

• Explore all flow cytometry controls
• Learn how to build accurate gating strategies
• Titrate your antibodies for better signal