Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging methods

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.

Automated summary

This article is part of a series providing advanced protocols for the study of dendritic cells (DC). It includes procedures for analyzing DC phenotypes, generating DC from various tissues, and studying DC functions using fluorescence microscopy. The protocols cover various methods such as intravital microscopy, multiparameter fluorescence microscopy, and 3D cell culture models. The article has been peer-reviewed by experts and is considered an essential resource for basic and clinical DC immunologists.