Development, application and computational analysis of high-dimensional fluorescent antibody panels for single-cell flow cytometry

Jolanda Brummelman, Claudia Haftmann, Nicolás Gonzalo Núñez, Giorgia Alvisi, Emilia M C Mazza, Burkhard Becher, Enrico Lugli

Research output: Contribution to journalArticlepeer-review


The interrogation of single cells is revolutionizing biology, especially our understanding of the immune system. Flow cytometry is still one of the most versatile and high-throughput approaches for single-cell analysis, and its capability has been recently extended to detect up to 28 colors, thus approaching the utility of cytometry by time of flight (CyTOF). However, flow cytometry suffers from autofluorescence and spreading error (SE) generated by errors in the measurement of photons mainly at red and far-red wavelengths, which limit barcoding and the detection of dim markers. Consequently, development of 28-color fluorescent antibody panels for flow cytometry is laborious and time consuming. Here, we describe the steps that are required to successfully achieve 28-color measurement capability. To do this, we provide a reference map of the fluorescence spreading errors in the 28-color space to simplify panel design and predict the success of fluorescent antibody combinations. Finally, we provide detailed instructions for the computational analysis of such complex data by existing, popular algorithms (PhenoGraph and FlowSOM). We exemplify our approach by designing a high-dimensional panel to characterize the immune system, but we anticipate that our approach can be used to design any high-dimensional flow cytometry panel of choice. The full protocol takes a few days to complete, depending on the time spent on panel design and data analysis.

Original languageEnglish
Pages (from-to)1946-1969
Number of pages24
JournalNature Protocols
Issue number7
Publication statusPublished - Jul 2019


  • Algorithms
  • Antibodies, Monoclonal
  • Biomarkers
  • Buffers
  • Coloring Agents
  • Computational Biology/methods
  • Flow Cytometry/methods
  • Fluorescent Antibody Technique/methods
  • Forkhead Transcription Factors/immunology
  • Humans
  • Signaling Lymphocytic Activation Molecule Family/immunology
  • Single-Cell Analysis/methods
  • T-Lymphocytes/cytology


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