The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

Fluorochrome, antibodies and detectors are important. The journey of a thousand cells starts with a good fluorescent panel. The polychromatic panel is the combination of antibodies and fluorochromes. These will be used during the experiment to answer the biological question of interest. When you only need a few targets, the creation of the panel is relatively straightforward. It’s only when you start to get into more complex panels with multiple fluorochromes that overlap in excitation and emission gets more interesting. 

FLUOROCHROMES

Both full spectrum and traditional fluorescent flow cytometry rely on measuring the emission of the fluorochromes that are attached to the antibodies of interest. But could be measuring some biological process like calcium flux or the cell cycle. With each fluorochrome, there are three things to keep in consideration as you choose them.

1. Excitation Profile

Based on the excitation lasers you have on your instrument will help determine which fluorochromes you can possibly use. However, a second consideration is the secondary laser lines that can excite the fluorochrome of interest. In figure 1, below, you can see three different fluorochromes, PE (top), PE-Cy5 (Middle) and PerCP-Cy5.5 (Bottom). Excitation curves are dotted and emission curves are filled in. Five common laser lines are highlighted as well. 

Figure 1: Excitation and emission profiles of three common fluorochromes. 

Looking at the PE trace, you can see that the PE molecule has an excitation at 488 and 561 nm. Historically, we used the 488 laser to excite PE. But with the introduction of the 532 and 561 nm lasers, we are able to get better excitation. This becomes an issue  if you have a detector off the 488 nm laser. As the PE signal can be measured there, which is a real signal, and can impact compensation. 

Moving to the PE-Cy5 dye, you can see the excitation profile of PE. But to the left, there is an excitation peak. therefore, if you were to use this dye, you would measure it off the 561 excitation. But could also see the Cy5 excitation in the red laser. Be careful while using this dye. Not only  because of the potential degradation of the tandem, but also as the excitation of it is independent of the acceptor excitation. 

These factors get even more complicated if we look at PerCP-Cy5.5.  While we usually measure this dye off the 488 nm laser, notice there is a robust excitation at 405 nm. Now, turning our attention to the tandem partner of this dye.  There is an excitation at 633 nm, and while only about 28% of maximum, means that it is possible to measure the tandem independent of the acceptor dye. Clearly this dye can cause issues all over the spectrum. 

2. Emission Profile

Looking at  the emission profiles for a minute, figure 2 indicates the emission of several PE tandem dyes. Also, the corresponding filters used to measure these dyes. 

From these profiles, it is clear that there may be problems with certain combinations. For example PE-Cy5 and PE-CY5.5 have a lot of overlap that can result in potential issues in the panel. PE-Cy7 shows a small emission from the donor (PE).

Figure 2:  Emission profile of PE and PE-tandems with common filter ranges to measure the emission

This is due, in part, to the fact that the Cy-7 excitation doesn’t overlap the PE emission very well. 

3. Relative Brightness

If we label the same antibody with different fluorochromes. It is possible to measure the relative brightness of the fluorochromes and generate a chart like this one. 

Table 1:  Relative brightness table of some fluorochromes on two different instruments.

Cells stained with the same antibody labeled with different fluorochromes and run on either a 3-laser (405, 488, 633) or a 4-laser (405, 488, 532, 633) instrument. Then calculate the staining index. Relative brightness of the fluorochrome ranked using the 3-laser system. 

Relative brightness is impacted by the excitation source, as well as the filter range for each detector.  For example, AlexaFluorⓇ 532 (AF5632). On the three laser system, with a suboptimal excitation the SI is only ~12, while when the fluorochrome is maximally excited, the SI jumps up to ~92. 

While you can find the brightness charts online, this isn’t a hard experiment to perform. So, having one for your instrument configuration (Laser and laser power, filters, etc..) is not a bad idea.

Figure 3:  Excitation and emission profile of AlexaFluorⓇ 532

ANTIBODIES

It goes without saying that the antibodies are an important component of the panel design process. So, without the correct antibodies, the panel is worthless. But in thinking about antibodies, there are several considerations that should be addressed as the panel is designed. 

1. Correct Clone 

Different clones can bind to different parts of the target antigen. Depending on the question being asked, the clone may be more or less important. You can find this information in the literature. If you’re in doubt, I like to recommend benchsci.com as a resource. You can search their database using target, cell type, assay type and more. The system will return figures from publications, allowing you to make a more informed decision. 

2. Co-expression  

If two or more markers are expressed on the same cell, this leads to a loss of resolution with certain fluorescent pairs. So it’s important to start by drawing out your strategy to identify your cells of interest. One such method is shown here, which was taken from this paper by Macirorowski et al (2017)

Figure 4:  Method to determine marker co-expression on target cells

A hierarchical structure is developed to determine which markers may be co-expressed. As seen in panel B, the Treg cells co-express CD3, CD4 and CD25. BD bioscience terms this the Resolution Impact, and have published a chart showing some common fluorochrome pairs. 

Figure 5: RIM chart from BD Biosciences

This information is useful to determine what pairs not to use. 

3. Antigen density

Traditional panel design teaches that we pair bright fluorochromes with low expression targets. So, for many years, this was a best guess based on the literature. Fortunately, there are now two datasets that researchers can use to help sort out these issues. 

  • Kalina et al. (2019) PMID: 31708916 -Used fluorescence data for characterization
  • Amir et al. (2019) PMID:31244854 – Used CyTOF data for characterization

These datasets show the different immune subsets, allowing  the researcher to check the specific expression patterns of their antigen of interest.

DETECTORS

The third component to building a good polychromatic panel is to know the sensitivity of your detectors. So, the idea here is that each detector is going to receive some signal from the other fluorochromes in the panel. Therefore, this signal can reduce the sensitivity of the detector, making it harder to identify the positive from dim from negative. The most common way to do this is to calculate the spillover spreading matrix (SSM). 

Figure 6: SSM of a simple panel calculated using FCS Express

The above figure (Fig 6) shows a simple SSM, as calculated using FCS Express. Flowjo has a similar capability. In both cases, this can be calculated from your single stain controls. 

The way to interpret this figure is how much a given fluorochrome (rows) can impact the detector (columns). Color-coded to identify those combinations that may be less ideal – take APC-CY7 into APC, we see that has the highest value in the chart. Then this can be interpreted that this is not a good combination if you want to make a sensitive measurement in the APC detector. Look down a detector. The APC detector is impacted the most by this spillover. While PerCP is a culprit of causing the greatest overall loss of sensitivity.

CONCLUSION

Gather this information. Then start building a polychromatic panel. Identify the most important antigens (those targeting the information you need to collect). Then pair those with the brightest fluorochrome. Ensure to minimize the resolution impact and avoid detectors and or fluorochromes that have a high value in the SSM. 

If all that fails, there is always the OMIP. Good luck and happy designing!

To learn more about important control measures for your flow cytometry lab, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

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Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

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