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Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.