Tools to Improve Your Panel Design – Determining Antigen Density

When a researcher chooses to use flow cytometry to answer a scientific question, they first have to build a polychromatic panel that will take advantage of the power of the technology and experimental design.

When we set up to use flow cytometry to answer a scientific question, we have to design a polychromatic panel that will allow us to identify the cells of interest – the target of the research.  To identify these cells, we need to build a panel that takes advantage of the relative brightness of the fluorochromes, the expression level of the different proteins on the cell, and the performance of the instrument, among other things. 

In its most basic form, the goal is to maximize the sensitivity of the measurement on the target cells.  We achieve this by pairing brighter fluorochromes with lower expressed targets, while minimizing the loss of resolution in the target channels.

One of the first steps in the process is to rank the expression level of the targets on the cells from the major sub-setting markers (CD3, CD4, CD19, etc.) to the targets of our investigation. To determine the answer to these questions, we need to research the literature.  Fortunately, there are several resources available to help with this work. 

The first is the website Benchsci.com. This website uses AI to curate published papers, identifying targets, clones, cells, assays, and more.  When you search for a given target, you are able to see the data where it was used, which allows you to make a more informed decision as to the utility of the target. Benchsci.com also introduced a tool that takes advantage of their AI to help select reagents for your research.

The second resource is a publication by Kalina and colleagues (2019).  In this paper, the authors measured the expression levels of CD antigens from 1 to 100 on 47 immune cell subsets. They used PE-labeled antibodies to calculate expression levels. All the data are accessible at www.hcdm.com. Example data, providing an overview of the expression levels are shown in Figure 1. 

Figure 1: Expression pattern of 29 CD antigens on the surface of immune cell subsets (across the top).  The color indicates the intensity of expression, while the size of the circles represents the frequency of the population in the given subset.

The third resource is a paper by Amir and colleagues (2019). They used mass cytometry to do the same thing that Kalina and colleagues did using fluorescent tags. In this paper, the authors screened 350 antibodies and used the automated analysis platform Astrolabe. The data, which will allow the researcher to explore their target antigens, are available at this site.

Figure 2: Expression pattern of different antigens in various cellular subsets (horizontal). The color represents the intensity of the signal, and the size of the circle represents the percent positive population.

Previously, determining the expression level of different antigens was a combination of guesswork and detective work. You had to read multiple journals to get an idea of what others had published, this only got more complex and time consuming when antigens started to have multiple clones. Enter automation and Artificial Intelligence in the guise of Benchsci.com. This tool is an excellent resource to help researchers identify the proper clone to use for their work.

Additionally, two groups used different approaches to provide the research community with the tools to determine the antigen density on different cellular subsets. Both have made their data publicly available so that the research community can take full advantage of these resources.

Thus, the first step of panel design has gone from frustrating and annoying to a simple matter of turning your web browser to three sites to get all the information you need. In the next blog on panel design, we’ll discuss the similarities and differences between panel design for traditional fluorescent flow cytometry and spectral cytometry, with a dash of mass cytometry thrown in for good measure.

Join Expert Cytometry's Mastery Class
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.

Similar Articles

The Essential Dos and Don'ts of NGS (Next Generation Sequencing)

The Essential Dos and Don'ts of NGS (Next Generation Sequencing)

By: Deepak Kumar, PhD

Next Generation Sequencing (NGS) is a rapidly evolving and widely used method worldwide in both academic and non-academic settings. One of the most valuable aspects of NGS is producing millions of sequenced reads with diverse read lengths from small amounts of input DNA. NGS methods are extremely versatile; producing reads as short as 75 bp, as seen in SOLiD sequencing, to long reads ranging upwards of 1000bp in the case of Pyrosequencing.  Both long and short reads fill a unique niche for researchers. Longer reads generated from NGS are excellent for genomic rearrangement and genome assembly projects; especially when there…

This Is How Full Spectrum Cytometry Works

This Is How Full Spectrum Cytometry Works

By: Tim Bushnell, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…

My Proven 5-Point Fast Track To A Career In Flow

My Proven 5-Point Fast Track To A Career In Flow

By: Tim Bushnell, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…

Up Your Stain Game With These 7 Non-Fluorescent Histology Dyes

Up Your Stain Game With These 7 Non-Fluorescent Histology Dyes

By: Heather Brown-Harding, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…

3 Ways Flow Cytometry Can Be Used To Research Bacteria

3 Ways Flow Cytometry Can Be Used To Research Bacteria

By: Tim Bushnell, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…

Avoid Flow Cytometry Faux Pas: How To Set Voltage The Right Way

Avoid Flow Cytometry Faux Pas: How To Set Voltage The Right Way

By: Tim Bushnell, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…

Discover The Myriad Applications Of Beads In Flow Cytometry

Discover The Myriad Applications Of Beads In Flow Cytometry

By: Tim Bushnell, PhD

What is the single-most important feature of a flow cytometry experiment? Arguably, it’s the stained cells that gather data about biological processes of interest. However, a flow cytometer can measure cell-like particles as well as cells, which opens the realm of cytometry to the use of microspheres. Most researchers are familiar with the 4-Cs that beads can be used for: Control, Calibration, Compensation, and Counting. Beyond the 4-Cs, many are familiar with the multiplex bead assays for measuring analytes. Today, we will take a look beyond these well-known uses and discover the myriad applications of the “Mighty Microspheres.”

Mass Cytometry Revolves Around These 5 Things

Mass Cytometry Revolves Around These 5 Things

By: Tim Bushnell, PhD

Because mass cytometry allows users to characterize masses so effectively, data can be normalized much more efficiently than what traditional fluorescent flow will permit. If there is no working CyTof at your institution, you can still partner with CyTof-friendly research institutions that have the technology on hand. And because the samples are fixed, you can ship them overnight. This way, they will be analyzed for you. Today’s article will summarize the functionality of mass cytometry technology. This tech has been commercialized largely by Fluidigm in the CyTof systems. There are 5 key points to cover, or takeaways, that cytometrists should…

3 Ways To Improve Flow Cytometry Troubleshooting

3 Ways To Improve Flow Cytometry Troubleshooting

By: Tim Bushnell, PhD

A lot of the troubleshooting is focused on fluidics issues. If you sit down and think about your workflow, and how you might want to add a couple of little tweaks here and there which will ultimately help you improve the quality of your data as well as aid you in identifying issues before they become problems your troubleshooting will be much smoother. Consider these three things, what do you before you start collecting data, ensure you have appropriate plots of time vs fluorescence for each of the lasers your using and apply appropriate gating procedures.

Top Technical Training eBooks

Get the Advanced Microscopy eBook

Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.