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Updated: Jan 23, 2026
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Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery
Published on: October 4, 2018
21.8K
Development, application and computational analysis of high-dimensional fluorescent antibody panels for single-cell
Jolanda Brummelman1, Claudia Haftmann2, Nicolás Gonzalo Núñez2
1Laboratory of Translational Immunology, Humanitas Clinical and Research Center, Rozzano, Milan, Italy.
Nature Protocols
|June 5, 2019
Summary
This study simplifies 28-color flow cytometry panel design by providing a reference map for fluorescence spreading errors. This advance accelerates high-dimensional single-cell analysis, particularly for immune system characterization.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Single-cell analysis is revolutionizing biological research, especially immunology.
- Flow cytometry offers high-throughput single-cell analysis and has been extended to 28 colors.
- Limitations of flow cytometry include autofluorescence and spreading error (SE), hindering dim marker detection and panel design.
Purpose of the Study:
- To describe the steps for achieving 28-color flow cytometry measurement capability.
- To provide a reference map of fluorescence spreading errors in a 28-color space.
- To offer detailed computational analysis instructions for complex flow cytometry data.
Main Methods:
- Development of a 28-color antibody panel.
- Creation of a fluorescence spreading error reference map.
- Application of computational algorithms (PhenoGraph, FlowSOM) for data analysis.
Main Results:
- Successful implementation of 28-color flow cytometry.
- A comprehensive reference map simplifying panel design and predicting antibody combination success.
- Detailed computational analysis protocols for high-dimensional data.
Conclusions:
- The described approach facilitates the design and analysis of complex, high-dimensional flow cytometry panels.
- This method streamlines the characterization of the immune system and other biological systems.
- The protocol is efficient, taking only a few days to complete.
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