Mass cytometry analysis of immune cells in the brain

Abstract

Immune cells comprise a diverse and dynamic cell population that is responsible for a broad range of immunological activities. They act in concert with other immune and nonimmune cells via cytokine-mediated communication and direct cell–cell interactions. Understanding the complex immune network requires a broad characterization of its individual cellular components. This is especially relevant for the brain compartment, which is an active immunological site, composed of resident and infiltrating immune cells that affect brain development, tissue homeostasis and neuronal activity. Mass cytometry, or CyTOF (cytometry by time-of-flight), uses metal-conjugated antibodies to enable a high-dimensional description of tens of markers at the single-cell level, thereby providing a bird's-eye view of the immune system. This technique has been successfully applied to the discovery of novel immune populations in humans and rodents. Here, we provide a step-by-step description of a mass cytometry approach for the analysis of the mouse brain compartment. The different stages of the procedure include brain perfusion, extraction of the brain tissue and its dissociation into a single-cell suspension, followed by cell staining with metal-tagged antibodies, sample reading using a mass cytometer, and data analysis using SPADE and viSNE. This procedure takes <5 h (excluding data analysis) and can be applied to study modifications in the brain's immune populations under normal and pathological conditions.

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Figure 1: Brain compartment analysis steps.
Figure 2: Perfusion quality evaluation steps.
Figure 3: Preliminary gating of a brain compartment mass cytometry sample.
Figure 4: The naive brain immune compartment.

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Acknowledgements

We thank S. Schwarzbaum and S. Urim for editing the paper; M. Schwartz, S. Shen-Orr, T. Ben-Shaanan and M. Schiller for their advice and helpful remarks; and A. Grau, Y. Sakuory and the Biomedical Core Facility at the Technion Faculty of Medicine for technical support and comments. This study was supported by the Israeli Ministry of Science, Technology & Space (MOST; 3-12070), the Prince Center for Neurodegenerative Diseases, Israeli Society for Science (ISF; 1862/15), an FP-7-CIG grant (618654) and the ADELIS Foundation. A.R. is an International Howard Hughes Medical Institute (HHMI)–Wellcome Trust researcher.

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B.K. designed the protocol, acquired the data and wrote the manuscript; T.D. contributed to the development of the protocol and to writing of the manuscript; and A.R. led the experimental design and revised the manuscript.

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Correspondence to Asya Rolls.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gating strategy for flow cytometry analysis.

Cells from the (a) blood and (b) brain compartment are selected according to the forward scatter (FSC) and side scatter (SSC) area (A) parameters. Then, doublet exclusion is performed using height (H) versus width (W) parameters of FSC and SSC accordingly, and live/dead discrimination is applied using Zombie dye.

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Supplementary Figure 1 and Supplementary Table 1. (PDF 592 kb)

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Korin, B., Dubovik, T. & Rolls, A. Mass cytometry analysis of immune cells in the brain. Nat Protoc 13, 377–391 (2018). https://doi.org/10.1038/nprot.2017.155

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