Guidelines for mouse and human DC generation

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. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34(+) cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34(+) cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. 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 of guidelines on dendritic cells, providing advanced protocols for the preparation and characterization of murine and human dendritic cells. The protocols cover the preparation and generation of dendritic cells from different cellular sources, including the generation of different subsets such as cDC1, cDC2, and pDC using Flt3L in mice, and the generation of bone marrow-derived dendritic cells using GM-CSF. For human dendritic cells, the protocols focus on the culture of CD34(+) cells on OP9 cell layers to generate different subsets, as well as the generation of dendritic cells from peripheral blood monocytes. The article has been peer-reviewed and approved by the co-authors, making it an essential resource for basic and clinical dendritic cell immunologists.