Designing a multicolor flow cytometry panel

A quick and easy guide to help you build multicolor panels for flow cytometry. 

Published July 21, 2023.

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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.

Once you know which antigens you want to study, you need to build your panel. 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

You will only be able to use fluorophores excited by the corresponding wavelength of light from the laser (Figure 1).

Figure 1. Common lasers used in flow cytometry.

The emission wavelength of your fluorophores should also be matched to the available filters and the wavelengths they allow to pass. The relative brightness of fluorophores and their excitation and emission spectra can be checked using our fluorophore chart.

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. 

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 2) and dimmer fluorophores to detect higher abundance targets.  

Figure 2. An antigen expressed at a 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 3). Compensation can be used to control the effects of spectral overlap (see below), but it is worth sacrificing some brightness in one detector to avoid spillover (Table 1).

Figure 3. Where possible, choose fluorophores with little or no overlap in their emission spectra.

Table 1. 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 fluorophore being measured².

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 4).

Figure 4. 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 5).

Figure 5. 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. There are some useful 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 or 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.

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