High-Dimensional Immunophenotyping with Fluorescence-Based Cytometry: A Practical Guidebook

  • Florian MairEmail author
  • Aaron J. Tyznik
Part of the Methods in Molecular Biology book series (MIMB, volume 2032)


Recent technological advances have greatly diversified the platforms that are available for high-dimensional single-cell immunophenotyping, including mass cytometry, single-cell RNA sequencing, and fluorescent-based flow cytometry. The latter is currently the most commonly used approach, and modern instrumentation allows for the measurement of up to 30 parameters, revealing deep insights into the complexity of the immune system.

Here, we provide a practical guidebook for the successful design and execution of complex fluorescence-based immunophenotyping panels. We address common misconceptions and caveats, and also discuss challenges that are associated with the quality control and analysis of these data sets.

Key words

Immunology High-dimensional Fluorescence Flow cytometry Polychromatic Panel design Spillover spreading SSM Compensation Staining 



The authors thank all members of the Prlic lab and Dr. Sabine Spath for critical reading of the manuscript. F.M. and A.J.T. are supported by a Marylou scholarship from the International Society for Advancement of Cytometry (ISAC).


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Vaccine and Infectious Disease DivisionFred Hutchinson Cancer Research CenterSeattleUSA
  2. 2.BD BiosciencesLa JollaUSA

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