本文是树突状细胞指南系列文章的一部分,它提供了一系列最先进的准备方案,通过流式细胞术进行表型分析,代,荧光显微镜,以及来自淋巴器官和各种非淋巴组织的小鼠和人树突状细胞(DC)的功能表征。最近的研究已经提供了越来越多的表型不同的常规DC(cDC)子集的证据,一方面表现出一定的功能可塑性。但另一方面,它们的特点是依赖于组织和上下文的功能专业化。这里,我们描述了用于小鼠和人cDC的功能表征的选择。前两个协议说明cDC内吞和代谢的分析,随后是cDC群体的转录组学和蛋白质组学表征指南。然后,一组更大的测定描述了体外cDC迁移的表征,离体,和体内。最终指南测量cDC炎性体和抗原(交叉)呈递活性。虽然所有协议都是由经验丰富的科学家编写的,他们在工作中经常使用它们,这篇文章也得到了领先专家的同行评审,并得到了所有合著者的批准,使其成为基础和临床DC免疫学家的重要资源。
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.