While flow cytometry is a powerful tool for identifying and analyzing multiple antigens simultaneously, increasing the number of antigens and fluorophores also increases the complexity of the experimental design. To address this, implementing a systematic workflow for multicolor flow cytometry is essential for ensuring accurate results and reproducibility.

Once you know which antigens you want to study, you need to build your panel. Flow cytometry panel design is a critical step that involves selecting markers optimized for the equipment intended, ensuring optimal performance and data quality.

Usually, this is the most challenging aspect of setting up a multicolor flow cytometry experiment. The steps below aim to provide a quick and easy guide to help you in the process¹.

Know your flow cytometer

Before designing your multicolor flow panel, you will need to determine the following factors:

1. The number and type of lasers

2. The number of detectors

3. The type of filters available on your flow cytometer

It is important to configure your panel for the specific equipment intended for your experiment, taking into account the spectral properties of your flow cytometry instrument. Understanding these spectral properties helps optimize panel design and minimize spectral overlap.

You will only be able to use fluorophores excited by the corresponding wavelength of light from the laser (Figure 13).

Figure 13. Common lasers used in flow cytometry

You should match the emission wavelength of your fluorophores to the available filters and the wavelengths they allow to pass. You can check the relative brightness of fluorophores and their excitation and emission spectra using our fluorophore chart. Substantial variation can exist between different flow cytometers, so it is important to tailor your panel design to the specific flow cytometry instrument being used.

Refer to your instrument’s manual or speak to your core facility manager to ensure optimal detection.

Know your cell populations, antigens, and fluorophores

Some cell populations are rare, or antigen density is low due to functional differences and cell activation levels. It is important to distinguish between different cell types, such as myeloid lineage, T cells, and malignant cells, and to analyze the internal complexity within the same cell to accurately characterize each cell population.

A highly expressed antigen will usually be detected and resolved from the negative control with almost any fluorophore. Generally, when designing a multicolor flow cytometry panel, use the brightest fluorophores (such as PE or APC) for low or unknown antigen expression targets or rare cell populations (Figure 14) and dimmer fluorophores to detect higher abundance targets. This approach is essential for accurately identifying cellular subpopulations and immune cells within each cell population.

Figure 14. An antigen expressed at lower density might require a higher signal-to-background ratio provided by a brighter PE or APC conjugate to separate positive cells adequately from unstained cells.

Bear in mind that other factors, such as sample buffer pH, can affect fluorophore brightness. The relative fluorophore intensity also depends on the instrument due to differences in the laser and filter combinations. Be sure to use the appropriate FACS instrument for the fluorophores you wish to detect.

Minimize spectral overlap

For best results with multicolor flow cytometry, choose the brightest fluorophores with little or no overlap between their emission spectra (Figure 15). When using multiple fluorochromes and tandem dyes in complex panels, it is essential to understand their spectral properties to optimize compensation and minimize spectral overlap.

You can use compensation to control the effects of spectral overlap (see below), but it is worth sacrificing some brightness in one detector to avoid spillover (Table 3). Selecting fluorophores that emit at a longer wavelength can help reduce spectral overlap, and the emitted light from each fluorophore is detected and measured to analyze cell characteristics.

Figure 15. Where possible, choose fluorophores with little or no overlap in their emission spectra.

Table 3. Examples of good and poor fluorochrome combinations for flow cytometry.

Fluorescence compensation

Fluorophores often have some overlap in their emission spectra, as demonstrated in the example below for PE and FITC. This overlap can generate a false positive signal as fluorescence from more than one fluorophore may be detected in a single channel. Spectral overlap can be problematic in multicolor experiments. Therefore, it must be corrected using compensation to ensure that the fluorescence detected is genuinely derived from the measured fluorophore².

Improper compensation can lead to analytical errors in multicolor flow cytometry experiments, affecting data accuracy and reliability.

Before compensation

Following excitation with the blue laser (488 nm), FITC emission is primarily detected in the channel-specific for FITC, but the emission tail lies within the range of the filter used to detect PE. These are seen as false positive signals in the PE channel, meaning that cells positive for FITC will appear positive for PE (Figure 16).

Figure 16. False-positive PE signals generated by FITC due to spectral overlap

After compensation

To compensate, a sample stained only with a FITC-labeled antibody is required. The settings can then be adjusted until no FITC signal is seen in the PE channel. Following compensation, FITC emission is solely detected in the channel specific for FITC (Figure 17).

Figure 17. Compensation removes false positive signals caused by spectral overlap.

Hints and tips for applying compensation in multicolor flow cytometry

The procedure for setting the correct fluorescence compensation follows the same basic principles on any cytometer. However, due to subtle differences between instruments, we always recommend reviewing the manufacturer’s instructions. Quality control and a systematic workflow are essential for obtaining optimal results, as they help address substantial variation between instruments and ensure consistency in immune profiling experiments. These are some valuable principles to follow:

• Some fluorophore combinations should be avoided (eg APC and PE-Cy5) due to the high degree of emission overlap

• Generally, compensate with the fluorophores from the far-red end of the spectrum (higher wavelength) stepwise down to the lower end of the spectrum. Do not forget to check the compensation in all channels

• Avoid fluorophore combinations where a bright fluorophore can spill over significantly into a channel if attempting to detect a dimmer population

• Compensation is correctly set when the median of the negative population is equal to the median of the positive population in the spillover channel

Compensation controls are required for each fluorophore and should contain a positive and negative population. The following guidelines should be applied:

• These controls should only be used to set compensation

• The fluorophore used as a compensation control must match the fluorophore used in the experiment

• The positive population should be at least as bright, if not brighter, than any sample the compensation will be applied to

• The positive population should form at least 10% of the total sample population

• The background fluorescence (autofluorescence) of the positive population should be the same as the negative control

• Ideally, the positive and negative populations in the control samples should be the same type of cell. If this is not possible, consider the use of compensation beads. Alternatively, different cells for your compensation controls can be used if they express the markers of interest

• If a marker of interest is rare or possibly absent in the control cells, a different antibody directed against a more common marker can be used if it carries the same fluorophore

Obtaining optimal results depends on rigorous quality control and a systematic workflow to address such limitations as substantial variation between instruments and the complexity of multicolor flow cytometry.

References

1. Busuttil Crellin, X.,, McCafferty, C.,, Van Den Helm, S.,, et al. Guidelines for panel design, optimization, and performance of whole blood multicolor flow cytometry of platelet surface markers Platelets  31 (7),845-852 (2020)
2. Szaloki, G.,, Goda, K. Compensation in multicolor flow cytometry Cytometry A 87 (11),982-985 (2015)