Abstract
Dendritic cells (DCs) encompass several subsets that are essential for shaping immune responses. Here, we present a protocol for ex vivo functional analysis and purification of DC subsets from blood and secondary lymphoid organs using flow cytometry. We describe the steps for isolating mononuclear cells from blood, lymph nodes, and spleen. We then detail the procedures for phenotypic characterization of DC subsets, intracellular staining to assess their cytokine production, and fluorescence-activated cell sorting (FACS) to isolate individual DC populations. For complete details on the use and execution of this protocol, please refer to Gardet et al.1.