Skip to main content Skip to navigation

Flow Cytometry Panel Design

 

Overview
Down Arrow Up Arrow

Multicolor flow cytometry is a powerful technology that enables the simultaneous analysis of multiple markers at the single-cell level. With the increase in the number of detectable parameters, the design of a multicolor flow cytometry panel can be challenging and requires an understanding of several factors that can influence panel performance:

  • Biology—Antigen density and co-expression
  • Fluorochrome—Brightness and spillover
  • Instrument—Configuration and set-up


In the following series of videos, you can learn the essential principles of flow cytometry panel design from BD Biosciences experts. The antibody panel design process begins with the identification of a biological hypothesis and is followed (in order) by the selection of the markers to be detected, understanding the settings on your flow cytometer, assigning the right fluorochromes to the identified markers, and then reviewing your panel. 

You can also use tools like OMIP or Optimised Multicolour Immunophenotyping Panels, to check whether a flow panel that may meet your requirements already exists.

performance1

Step 1

Define your experimental hypothesis

Defining your experimental hypothesis is the first step in flow cytometry panel design. Start with identifying:

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

 

The biological information that is desired is crucial as it dictates every other aspect of the flow cytometry experiment detailed in the coming steps. 

It is always useful to learn all about the biology of your target cell populations and their markers from other sources, before embarking on an attempt to design your flow cytometry panel.


Watch the flow cytometry panel design step 1 video to begin your panel design journey.

 
performance1

Step 2

Select the right marker

During the second step of the panel design process, you will 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 density, often defining lineages
  • Secondary antigen: Often expressed over a continuum
  • Tertiary antigen: Critical markers expressed at low density
  • Marker co-expression, especially of dim markers
  • The gating strategy needed to identify the population(s) of cells you wish to interrogate

 

Identifying the critical population will allow you to assign the fluorochromes with exceptional resolution to high-value markers that are instrumental in defining these critical populations. This will also enable you to map out the path towards detecting and analysing the critical populations using all the markers you have in your flow cytometry panel.


Watch the flow cytometry panel design step 2 video to understand the intricacies in identifying critical populations and selecting the markers for analysis.

 
performance1

 

 

 

Step 3

 

Know your flow cytometer

Knowing your instrument is essential. Understanding your instrument configuration will let you know how many markers and which fluorochromes your instrument can detect.


Elements to consider include:
 

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


It is often said that the first step in learning to love others is to understand them. If you love flow cytometry, you must first understand your flow cytometer and its capabilities.


You need to know which flow cytometer to use, which has the right lasers and can detect all the parameters you need for the markers you wish to study. The fluorochromes you can use in your panel depend on the lasers and filters that are available on your flow cytometer, making this understanding vital for panel design.


Watch the flow cytometry panel design step 3 video to understand and maximize the capabilities of your flow cytometer.

 
performance1

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
 
performance1

Step 5

Review your panel

Review your panel design and begin ordering your reagents. Remember to titrate your mass size reagents and optimize your staining protocol. Include proper controls for compensation, FMO and biological controls to help ensure optimal panel performance.

 

To find more panel design tips and some fun facts, download the flow cytometry panel design journey infographic.

Download Now
 
performance1
Videos
Down Arrow Up Arrow

Avoiding Resolution Loss When Using Fix and Permeabilization Buffers for Intracellular Staining

In this video you will learn helpful strategies to help prevent loss of resolution due to intracellular fixation and permeabilization buffers.

 
performance1

Understanding and Managing Fluorescence Spillover

In this video you will learn how to manage fluorescence spillover to get the most out of your flow cytometry experiments.

 
performance1

Relationship Between Fluorescence Spillover and Spread

This video will help you understand the relationship between florescence spillover and spread to help you implement the right strategies to resolve your populations of interest.

 
performance1

Minimizing the Impact of Spread in Flow Cytometry Experiments

This video provides strategies to help minimize the impact of spread in flow cytometry experiments to help improve resolution.

 
performance1

Panel Design Workflow for Optimal Fluorochrome-Antigen Matching

This video provides simple strategies for fluorochrome-antigen matching to help prevent the introduction of spread and subsequent resolution loss.

 
performance1
BD Horizon™ Tour Series
Down Arrow Up Arrow

BD Horizon™ Tour Series

Achieve: Putting It All Together: Panel Design Principles for Surface and Intracelluar Staining

This webinar explores concepts, best practices and available tools to help you efficiently navigate multicolor flow cytometry.

 

Speaker

Andrew D. Bantly

Technical Applications Specialist

Watch Now
performance1
Sentinel Marker Approach to Multicolor Flow Cytometry: An Experimental Design that Uses Key Markers to Define Cell Subpopulations to Extend Instrument Capability

Discover a new way of looking at fluorochromes and markers that can maximize the number of parameters achievable on any instrument.

Speaker

Bob Balderas, MBA

VP Biological Sciences & VP Market Development

BD Life Sciences–Biosciences

Watch Now
performance1
Resolve: Managing Instrument Setup to Address Fluorescence Spillover and to Maximize Resolution

This webinar offers a comprehensive look at best practices for managing instrument set up to help you maximize your instrument’s parameters while minimizing spillover and resolution loss. 

Speaker

Alan Stall

Principle Scientist 

BD Life Sciences–Biosciences

Watch Now
performance1
Elevate: The Importance of Surface Antigen Density and Intracellular Epitope Availability in Choosing the Appropriate Fluorochrome/Antibody Combination

This webinar explores the importance of understanding antigen density (the number of cell surface receptors on a cell) in relation to antibody/fluorochrome combinations for optimal panel design. 
 

Speaker 

Bob Balderas, MBA

VP Biological Sciences & VP Market Development

BD Life Sciences–Biosciences

 

Watch Now
performance1
Reveal: Implications of Relative Fluorochrome Brightness in Resolving the Population of Interest

This webinar explores the journey to finding what nature is hiding using flow cytometry by understanding the biology of your cells and the relative brightness of fluorochromes. 

Speaker

Bob Balderas, MBA

VP Biological Sciences & VP Market Development

BD Life Sciences–Biosciences

Watch Now

Watch Now
performance1
BD Harmony Series
Down Arrow Up Arrow

BD Harmony Series

Ask the Experts—A Solid Foundation for Simpler and Smarter Panel Design

This webinar offers viewers practical strategies for designing optimized multicolor flow cytometry panels by addressing frequently asked questions. 
 

Speakers

Bob Balderas, MBA

VP Biological Sciences & VP Market Development

BD Life Sciences–Biosciences


Mirko Corselli, PhD

Global Scientific Content Manager

BD Life Sciences–Biosciences


Rui Gardner, PhD

Head of Flow Cytometry Core Facility

Memorial Sloan Kettering Cancer Center


Yolanda Mahnke, PhD

Founder

FlowKnowHow LLC

Watch Now
performance1
Providing a Solid Foundation for Simpler Smarter Flow Cytometry Panel Design: Harmony Seminar Series

This webinar is part of the Harmony Series and offers viewers panel design best practices, with emphasis on spillover and identification of optimal fluorochrome combinations to simplify panel design.

Speaker

Mirko Corselli, PhD

Global Scientific Content Manager

Research Solutions, BD Life Sciences–Biosciences

Watch Now
performance1
A Solid Foundation for Simpler and Smarter Panel Design

This webinar explores strategies to help customers create a solid chromatic foundation for multicolor panel design
  

Speakers

Bob Balderas, MBA

VP Biological Sciences & VP Market Development

BD Life Sciences–Biosciences

Watch Now
performance1

Other

BD Panel Design Matters: Practical Aspects of Designing Flow Cytometry Panels

This webinar explores strategies for minimizing flow cytometry panel design pain points as well as panel optimization
 

Speaker

Alan Stall

BD Fellow, Principal Scientist, BD Life Sciences–Biosciences

Watch Now
performance1
Making Panel Design Easier in the Era of High-Dimensional Flow Cytometry

This webinar offers comprehensive strategies for adding efficiency to the flow cytometry panel design process

Speaker

Anis Larbi, PhD

Head of A*STAR Flow Cytometry Facility, Immuno-monitoring Platform and Principal Investigator, Biology of Aging Program 
Singapore Immunology Network (SIgN)

Watch Now
performance1
Compensation Matters: Considerations When Designing Your Flow Cytometry Panels

This webinar provides a robust overview on compensation best practices to help achieve your resolution goals. 

Speaker

Alan Stall

BD Fellow, Principal Scientist, BD Life Sciences–Biosciences

Watch Now
performance1
Resources
Down Arrow Up Arrow
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 

performance1

  

  

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

BD Flow Cytometers are Class 1 Laser Products