Skip to content

The Most Common Mistake Researchers Make When Designing Flow Cytometry Antibody Panels

Written by Tim Bushnell, PhD

Pairing highly expressed antigens (like CD3) with dimmer fluorochromes, and the antigens of interest with the brightest fluorochromes, is a key part of panel design with few tools to help.

With early generation instruments, this was relatively easy to determine, since fluorochrome choice was limited. With the advent of instruments capable of measuring more than 4 fluorochromes, there is a need to characterize the relative brightness of different fluorochromes under actual experimental conditions, rather than as free fluors.

Bigos et al (2004) first reported this in an abstract and it was later simplified in Maecker et al (2004). This equation (Figure 1) compares the  differences between the means of the positive and negative and corrects this value by dividing by two times the spread (as measured by the standard deviation) of the negative population.

This measure — the Staining Index (or SI) allows for the comparison of the relative brightness of fluorochromes.  As shown in Figure 2 below, if the different fluorochromes are attached to the same antigen, cells can be stained and the SI compared. Researchers who fail to consider SI values are making a significant mistake that can negatively impact their flow cytometry experiments. 

The above calculations reveals that PE > APC > FITC > PerCP   Thus, providing the researcher with critical information necessary to design the polychromatic panel.

There were some limitations to this Staining Index. Most notably was the fact that the data was corrected by the standard deviation of the noise. With many fluorochromes on digital instruments, at least part of the negative signal is background noise the can be attributed in part to the electronic noise in the flow cytometer. A modification of the SI was pulsed by Telford et al. (2009).  The equation for this correction is shown in Figure 3.

In this equation the difference between the medians of the positive and negative populations and divides that by the right hand of the negative distribution, as reflected by the 84% of the negative minus the median of the negative. This Separation Index (or SI) provides a similar metric to the Bigos/Maecker Staining Index as shown in Figure 4.

Be it Staining or Separation, the SI is a critical parameter and the experiment is straightforward, and can be performed using any highly-expressed antigen that is available in many fluorochromes.

Tim Bushnell, PhD


Advanced Microscopy

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

Modern Flow Cytometry

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

Advanced 4-10 Color Compensation

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.

Top 40 Networking Scripts For PhDs

If you want to get replies from top employers and recruiters, this ebook is for you. These networking scripts will show you the exact words ...

Informational Interviews For PhDs

If you want to learn how to set up and execute informational interviews with PhDs working in industry, this ebook is for you. Here, you ...

Industry Resume Guide For PhDs

If you have a PhD and want to create the perfect industry resume to attract employers, this ebook is for you. Here, you will get ...

Top 20 Industry Jobs For PhDs

If you want to learn about the top 20 industry careers for PhDs regardless of your PhD background, this ebook is for you. Here, you ...

Salary Negotiation For PhDs

If you have a PhD and want to learn advanced salary negotiation strategies, this ebook is for you. Here, you will learn how to set ...

Top 20 Transferable Skills For PhDs

If you want to learn the top 20 transferable skills the industry employers ranked as most important for PhDs to include on their resumes and ...

Related Posts You Might Like

6 Areas Of Consideration For Flow Cytometry Cell Cycle Analysis

Written by Tim Bushnell, PhD As discussed previously, cell cycle assays require optimization of fixation and dye concentrations, but that is just the beginning. There ...
Read More

Why Cell Cycle Analysis Details Are Critical In Flow Cytometry

Written by Tim Bushnell, PhD The lifecycle of a cell can be described in stages. In diploid cells, much of the time they exist in ...
Read More

How To Choose The Correct Antibody For Accurate Flow Cytometry Results

Written by Tim Bushnell, PhD Next to the flow cytometer itself, the most important component of a flow cytometry experiment comes down to the antibodies. ...
Read More

5 Essential Beads For Flow Cytometry Experiments

Written by Tim Bushnell, PhD Flow cytometry is designed to measure physical and biochemical characteristics of cells and cell-like particles using fluorescence. Fundamentally, any single-particle ...
Read More

How To Use Flow Cytometry To Measure Apoptosis, Necrosis, and Autophagy

Written by Tim Bushnell, PhD “If one approaches a problem with order and method there should be no difficulty in solving it — none whatever.” ...
Read More

Flow Cytometry Protocols To Prevent Sample Clumping

Written By Tim Bushnell, PhD Good flow cytometry depends on a high-quality, single cell suspension. If the cells put through the instrument are not of ...
Read More