Guidelines for DC preparation and flow cytometric analysis of human lymphohematopoietic tissues

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. Within this article, detailed protocols are presented that allow for the generation of single cell suspensions from human lymphohematopoietic tissues including blood, spleen, thymus, and tonsils with a focus on the subsequent analysis of DC via flow cytometry, as well as flow cytometric cell sorting of primary human DC. Further, prepared single cell suspensions as well as cell sorter‐purified DC can be subjected to other applications including cellular enrichment procedures, RNA sequencing, functional assays, and many more. 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. Dendritic cells are major regulators of immune responses. We here provide detailed protocols for the preparation of single cell suspension from human lymphohematopoietic tissues. Further, we provide gating strategies for flow cytometric analysis as well as cell sorting of primary human lymphohematopoietic DC subpopulations.