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Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging
Felix Bayerl1, David A Bejarano2, Giulia Bertacchi3
1Institute of Molecular Immunology, School of Medicine, Klinikum Rechts der Isar, Technical University of Munich (TUM), Ismaninger Str. 22, Munich, Germany.
Insights
This guide details advanced microscopy techniques for studying dendritic cells (DC). It covers DC preparation, imaging, and functional analysis in various tissues, aiding immunologists in understanding immune responses.
Area of Science:
- Immunology
- Cell Biology
- Microscopy
Background:
- Dendritic cells (DC) are crucial immune cells involved in initiating adaptive immunity.
- Understanding DC spatial organization, migration, and communication within tissues is vital for dissecting immune responses.
- Existing protocols may not fully capture the dynamic nature and complex functions of DCs in situ.
Purpose of the Study:
- To provide state-of-the-art protocols for the comprehensive analysis of mouse and human dendritic cells.
- To detail multiparameter fluorescence microscopy methods for exploring DC spatial organization and function in diverse tissue environments.
- To enable the study of DC dynamics, T cell cross-talk, and interactions with other tissue cells.
Main Methods:
- Preparation and phenotype analysis of dendritic cells (DCs) from lymphoid and non-lymphoid organs.
- Multiparameter fluorescence microscopy for visualizing DC subsets and their functions in fixed tissue sections.
- Intravital microscopy for studying DC dynamics and T cell interactions in vivo.
- 3D cell culture models for analyzing DC interactions with tissue cells.
Main Results:
- Detailed procedures for advanced imaging techniques are presented.
- Methods allow for large-scale visualization, identification, and quantitative analysis of DC subsets.
- Protocols facilitate the study of DC migration, communication, and functional roles in immunity.
- The study enables exploration of DC interactions with tissue cells and T cells.
Conclusions:
- This article serves as an essential resource for basic and clinical immunologists studying dendritic cells.
- The provided protocols enhance the understanding of dendritic cell biology and their roles in immune regulation.
- Advanced microscopy techniques are critical for dissecting the complex spatial and functional aspects of dendritic cells in immunity.
Abstract:
This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Here, we provide detailed procedures for a variety of multiparameter fluorescence microscopy imaging methods to explore the spatial organization of DC in tissues and to dissect how DC migrate, communicate, and mediate their multiple functional roles in immunity in a variety of tissue settings. The protocols presented here entail approaches to study DC dynamics and T cell cross-talk by intravital microscopy, large-scale visualization, identification, and quantitative analysis of DC subsets and their functions by multiparameter fluorescence microscopy of fixed tissue sections, and an approach to study DC interactions with tissue cells in a 3D cell culture model. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.
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