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Updated: Oct 21, 2025
Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols
Published on: October 18, 2016
Mass cytometry profiling of human dendritic cells in blood and tissues
Marcela Alcántara-Hernández1, Juliana Idoyaga2
1Microbiology & Immunology Department and Immunology Program, Stanford University School of Medicine, Stanford, CA, USA.
Insights
This study presents an optimized mass cytometry protocol for analyzing rare human dendritic cells (DCs) in blood and tissues. The method enhances the characterization of these crucial immune cells, even in low numbers.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Mass cytometry enables in-depth protein profiling of immune cells.
- Analyzing minor immune cell populations, like dendritic cells (DCs), is challenging due to low cell numbers and mass cytometry's throughput limitations.
- Dendritic cells are critical for initiating and modulating immune responses.
Purpose of the Study:
- To provide an optimized, step-by-step protocol for characterizing human dendritic cell populations in blood and tissues using mass cytometry.
- To enable the analysis of both known and emerging dendritic cell subsets.
- To facilitate the study of dendritic cells in various biological contexts, including health, disease, and vaccination.
Main Methods:
- Development of a detailed protocol for single-cell suspension generation, sample enrichment, staining, acquisition, and data analysis.
- Incorporation of a barcoding strategy to reduce acquisition variability and enable analysis of low cell numbers (~20,000).
- Emphasis on negative selection techniques for immune cell enrichment to prevent activation of minor populations.
Main Results:
- The protocol is optimized for the characterization of human dendritic cells using mass cytometry.
- The barcoding option allows for reliable analysis of limited dendritic cell numbers.
- Negative selection effectively enriches for minor immune cell populations without activation.
Conclusions:
- This robust and reliable protocol facilitates the comprehensive analysis of human dendritic cells.
- The optimized method supports the study of dendritic cells in diverse conditions, including health, disease, and vaccination.
- The protocol addresses the challenge of analyzing low-abundance dendritic cell populations, advancing immunological research.
Abstract:
The immune system comprises distinct functionally specialized cell populations, which can be characterized in depth by mass cytometry protein profiling. Unfortunately, the low-throughput nature of mass cytometry has made it challenging to analyze minor cell populations. This is the case for dendritic cells, which represent 0.2-2% of all immune cells in tissues and yet perform the critical task of initiating and modulating immune responses. Here, we provide an optimized step-by-step protocol for the characterization of well-known and emerging human dendritic cell populations in blood and tissues using mass cytometry. We provide detailed instructions for the generation of single-cell suspensions, sample enrichment, staining, acquisition and data analysis. We also include a barcoding option that reduces acquisition variability and allows the analysis of low numbers of dendritic cells, i.e., ~20,000. In contrast to other protocols, we emphasize the use of negative selection approaches to enrich for minor populations of immune cells while avoiding their activation. The entire procedure can be completed in 2-3 d and can be conveniently paused at several stages. The procedure described in this robust and reliable protocol allows the analysis of human dendritic cells in health and disease and during vaccination.
