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

Dendritic cells (DCs) populate nearly all tissues and represent the central orchestrators of immunity. Here, we present a protocol for the mild but efficient preparation of single-cell suspensions from multiple murine tissues and the downstream analysis of the DC network via high-parameter flow cytometry. Additionally, we provide evaluation strategies that facilitate the stringent separation of the DC family from other myeloid cells, particularly macrophages and monocytes, and include an in-depth assessment of DC-intrinsic heterogeneity. For complete details on the use and execution of this protocol, please refer to Amon et al.1.

Clec12A, CD301b, and FcγRIIB/III define the heterogeneity of murine DC2s and DC3s.

Over the last decade, multiple studies have investigated the heterogeneity of murine conventional dendritic cells type 2 (cDC2s). However, their phenotypic similarity with monocytes and macrophages renders their clear identification challenging. By creating a protein atlas utilizing multiparameter flow cytometry, we show that ESAM+ cDC2s are a specialized feature of the spleen strongly differing in their proteome from other cDC2s. In contrast, all other tissues are populated by Clec12A+ cDC2s or Clec12A- cDC2s (high or low for Fcγ receptors, C-type lectin receptors, and CD11b, respectively), rendering Clec12A+ cDC2s classical sentinels. Further, expression analysis of CD301b, Clec12A, and FcγRIIB/III provides a conserved definition of cDC2 heterogeneity, including the discovery of putative FcγRIIB/III+ DC3s across tissues. Finally, our data reveal that cell identity (ontogeny) dictates the proteome that is further fine-tuned by the tissue environment on macrophages and dendritic cells (DCs), while monocytes and plasmacytoid DCs (pDCs) display subset intrinsic default settings.