The immune system comprises distinct functionally specialized cell populations, which can be characterized in depth by mass cytometry protein profiling. Unfortunately, the low-throughput nature of mass cytometry has made it challenging to analyze minor cell populations. This is the case for dendritic cells, which represent 0.2–2% of all immune cells in tissues and yet perform the critical task of initiating and modulating immune responses. Here, we provide an optimized step-by-step protocol for the characterization of well-known and emerging human dendritic cell populations in blood and tissues using mass cytometry. We provide detailed instructions for the generation of single-cell suspensions, sample enrichment, staining, acquisition and data analysis. We also include a barcoding option that reduces acquisition variability and allows the analysis of low numbers of dendritic cells, i.e., ~20,000. In contrast to other protocols, we emphasize the use of negative selection approaches to enrich for minor populations of immune cells while avoiding their activation. The entire procedure can be completed in 2–3 d and can be conveniently paused at several stages. The procedure described in this robust and reliable protocol allows the analysis of human dendritic cells in health and disease and during vaccination.

免疫系统由不同的功能特化细胞群组成，可以通过质谱流式细胞术蛋白质分析来深入表征。不幸的是，质谱流式细胞术的低通量性质使得分析小细胞群变得具有挑战性。树突状细胞就是这种情况，它占组织中所有免疫细胞的 0.2-2%，但执行启动和调节免疫反应的关键任务。在这里，我们提供了一个优化的分步方案，用于使用质谱流式细胞术表征血液和组织中众所周知的和新兴的人类树突状细胞群。我们提供有关单细胞悬浮液的生成、样品富集、染色、采集和数据分析的详细说明。我们还包括一个条形码选项，可减少采集变异性并允许分析少量树突状细胞（即约 20,000 个）。与其他方案相比，我们强调使用负选择方法来富集少数免疫细胞群体，同时避免其激活。整个过程可以在2-3天内完成，并且可以方便地在几个阶段暂停。这个强大而可靠的协议中描述的程序允许在健康和疾病以及疫苗接种期间分析人类树突状细胞。