Multicolor Flow Cytometry Panel Design

Multicolor flow cytometry enables the simultaneous analysis of multiple markers at the single-cell level. By using fluorochrome conjugated antibodies that target specific cellular markers, researchers can analyze various cell characteristics like surface antigens and intracellular proteins. The design of a flow cytometry panel can be challenging and requires an understanding of several factors that can influence panel performance such as the fluorochromes and the instrument being used.

Overview

Optimizing Your Flow Cytometry Protocol

Flow Cytometry can be a complex process, requiring specialized expertise. Particularly, developing an optimized panel design for protein analysis requires careful consideration of factors such as marker identification and fluorochrome selection. At BD Biosciences, we are committed to guiding you through every step of this intricate process, ensuring that you achieve reliable and meaningful results.

Doing spectral? Check out our spectral flow cytometry resources.

When building assays, we are presented with hundreds of fluorochrome options and their different properties can make selection feel overwhelming. Check out our Fluorochrome Performance Chart with a handy cheat-sheet to help you prioritise clean fluorochromes and simplify your panel design.

"With the advice of the BD support team and with some minor tweaks while titrating and testing the panels, we were able to set up and validate those panels in a short timeframe and we were very happy with the results. The studies in which the panels were used were published in 2022 (PMID 35838348) and 2024 (PMID 38548324)."

Dr. Carolien van de Sandt

Sanquin Research

Landsteiner Laboratory

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Panel Design Video Guide

Panel Design for Flow Cytometry

In the following series of videos, you can learn the essential principles of how to design a flow cytometry panel from BD Biosciences scientists. Developing a panel design for protein analysis involves several steps to ensure the selection of antibodies that specifically recognize the target antigens with high affinity and minimal cross-reactivity, and includes:

Identification of an experimental hypothesis
Selection of the markers to be detected
Understanding the setting on your flow cytometer
Assigning the right fluorochromes to the identified markers
Reviewing the panel

Tools like Optimized Multicolor Immunophenotyping Panels (OMIP) and the BD Panel Repository can also be used to check whether a flow panel that may meet your requirements already exists.

Step 1: Define your experimental hypothesis

To define your experimental hypothesis, you need to start by identifying the following:

The biological information you are trying to obtain
The population(s) of cells you wish to interrogate (i.e., the critical population)
Which and how many markers you need
Whether targets are found on the cell surface or intracellularly

It is important to fully understand the desired biological information as it dictates the subsequent steps needed for your flow cytometry experiment. Therefore, it is useful to learn all about the biology of your target cell populations and their markers from reputable sources before attempting to design your flow cytometry panel.

Step 2: Select the right marker

Next, you would need to identify which and how many markers you need to identify in the cell population of interest.

Pay attention to:

Marker expression levels
- Primary antigen: Expressed at high densities, often defining lineages or cell subsets (e.g., CD4)
- Secondary antigen: Often expressed over a continuum, provides nuanced information about cellular states and phenotypes (e.g., CD45RA)
- Tertiary antigen: expressed at low density but is often critical for identifying rare or specialized cell populations within the sample (e.g., CD25)
Marker co-expression, especially of dim markers
The gating strategy needed to identify the population(s) of cells you wish to interrogate

Step 3: Know your flow cytometer

Understanding your instrument configuration will let you know how many markers and which fluorochromes your instrument can detect. This understanding is vital for your panel design.

Elements to consider include:

Laser wavelength for excitation
Number of detectors for each laser
Filters available to detect the fluorochromes

Step 4: Assign the right fluorochromes to the markers

Carefully select fluorochromes to resolve markers at all expression levels and minimize spectral overlap.

Consider using tools like a fluorochrome resolution ranking and a spectrum viewer to help assess:

Cross laser excitation
Fluorochrome spillover

Remember to pair bright fluorochromes with low expressing antigens and dim fluorochromes with high expressors. Keep in mind that spread only impacts the resolution of coexpressed markers.

Consider using laser-specific fluorochromes to reduce the complexity of the compensation and unmixing processes.

Once you are done, review your panel design and begin ordering your reagents. Remember to titrate your mass size reagents and optimize your staining protocol. Include proper compensation control, fluorescence minus one (FMO) controls and biological controls to ensure optimal panel performance.

Your next steps

Discover insights, including thought leader opinions, breakthrough research and flow cytometry publication reviews. Visit our blog

Evaluate Your Panel Design

Resources

Tools

Interactive Human Cell Map Clone Comparison Tool BD^®Spectrum Viewer BD FACSelect™ Buffer Compatibility Tool BD^® Research Cloud FlowJo™ v10 Software BD Fluorochrome Performance Guide

Webinars

Achieve: Putting It All Together: Panel Design Principles for Surface and Intracelluar Staining Sentinel Marker Approach to Multicolor Flow Cytometry: An Experimental Design that Uses Key Markers to Define Cell Subpopulations to Extend Instrument Capability Resolve: Managing Instrument Setup to Address Fluorescence Spillover and to Maximize Resolution Elevate: The Importance of Surface Antigen Density and Intracellular Epitope Availability in Choosing the Appropriate Fluorochrome/Antibody Combination Reveal: Implications of Relative Fluorochrome Brightness in Resolving the Population of Interest Ask the Experts—A Solid Foundation for Simpler and Smarter Panel Design Providing a Solid Foundation for Simpler Smarter Flow Cytometry Panel Design: Harmony Seminar Series A Solid Foundation for Simpler and Smarter Panel Design BD Panel Design Matters: Practical Aspects of Designing Flow Cytometry Panels Making Panel Design Easier in the Era of High-Dimensional Flow Cytometry Compensation Matters: Considerations When Designing Your Flow Cytometry Panels

Blog Articles

BD Harmony Webinars by Bob Balderas, BD Biosciences How Dyes Evolved: The History of the Modern Flow Cytometry Dye Panel Discussion on Validating Assays in Flow Cytometry High-Dimensional Flow Cytometry for Improved Data Analysis FlowJo University – The One Stop for All Your FlowJo™ and SeqGeq™ Software Training Needs

Ebooks

33C SE Panel Sheet | Immunophenotyping and cell isolation with high-parameter spectral flow cytometry BD Leukocyte Panel Blocks

E-learning Courses

Flow Cytometry Basics

Publications

Mahnke YD, Roederer M. Optimizing a multicolor immunophenotyping assay. Clin Lab Med. 2007;27(3): 469–485, v. doi: 10.1016/j.cll.2007.05.002
Mair F, Tyznik A. High-dimensional immunophenotyping with fluorescence-based cytometry: a practical guidebook. Methods Mol Biol. 2019;2032:1-29. doi: 10.1007/978-1-4939-9650-6_1
Nguyen R, Perfetto S, Mahnke YD, Chattopadhyay P, Roederer M. Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design. Cytometry A. 2013;83(3):306-315. doi: 10.1002/cyto.a.22251
Roederer M. Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry. 2001;45(3):194-205. doi: 10.1002/1097-0320(20011101)45:3<194::aid-cyto1163>3.0.co;2-c

Terms and Conditions

Panel iterations must be verified by a BD Applications Specialist. Valid only on panels designed by BD Biosciences in the last 2 months. Swaps that constitute more than a 20% change of the total number of reagents must be approved by BD.

Recommendations given by representatives of BD in the context of the creation and setup of customer-specific applications and assays and the composition of antibody panels for use in flow cytometry assays are provided in a diligent manner. However, the Customer acknowledges that (i) such recommendations should not be treated as a substitute for the Customer’s own examination, (ii) BD does not make any promises nor guarantees that recommendations are accurate and complete and will meet the Customer’s present or future needs or will produce positive or specific results and (iii) Customer remains solely responsible for validating and deciding on any such recommendations.

BD, therefore, accepts no liability whatsoever in connection with any such recommendation and/or any results generated on the basis of such recommendations and, to the fullest extent permitted by law, expressly disclaims any and all warranties and any liability in this respect. In particular, the Customer assumes sole responsibility for the validation, use, selection, and suitability of the recommendations for its needs and objectives.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

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