Guidelines for mouse and human DC functional assays

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. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue‐ and context‐dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)‐presentation activity. 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.

Microenvironmental Regulation of Macropinocytosis Facilitates Extracellular Fluid Sampling

“…To uncover whether 3D microenvironments generally enhance macropinocytosis, we injected macrophages between an agarose layer and a coverslip 19 . In this reductionistic 3D-like environment, cells experience both a substrate below and on top, and switch to a 3D-like behavior 20 , while having less freely accessible cell surface than on 2D substrates.…”

Protocol for mapping the heterogeneous dendritic cell network across the murine tissue landscape via high-dimensional flow cytometry

“…However, prepared single cell suspensions can be used for additional downstream applications including cellular enrichment and sorting, functional assays, and sequencing procedures. 2 , 3 , 4 Finally, the provided analysis template can be transferred to other murine tissues such as the intestine and its associated lymphoid tissues. 3 , 4 …”

“… 2 , 3 , 4 Finally, the provided analysis template can be transferred to other murine tissues such as the intestine and its associated lymphoid tissues. 3 , 4 …”

Adaptive pathfinding by nucleokinesis during amoeboid migration

“…Under-agarose migration assays Under-agarose migration assays were performed as described previously (Clausen et al, 2022). In brief, 4% UltraPure agarose (Invitrogen) in sterile water was mixed with 55°C prewarmed phenol-free RPMI-1640 (Gibco) supplemented with 20% FCS and 1× Hanks buffered salt solution pH 7.3 in a 1:3 ratio resulting in a final agarose concentration of 1%.…”