Protocol for phenotyping and isolation of dendritic cell subsets from blood and lymphoid organs of non-human primates and humans by flow cytometry

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.

Keywords: Cell Biology; Cell isolation; Cell-based Assays; Flow Cytometry; Immunology; Molecular Biology; Single Cell.

Graphical abstract

Figure 1

Complete gating strategy for identifying DC subsets in NHP peripheral blood Representative gating strategies to identify DC subsets among PBMCs are shown using pseudocolor density plots and 5% colored contour plots. Each dendritic cell subset can be followed in the gating strategy: pDC (orange); pre-DC (pink); cDC1 (yellow); cDC2 (blue).

Figure 2

Complete gating strategy for identifying DC subsets in NHP secondary lymphoid organs Representative gating strategies to identify DC subsets among SMCs (A) and LNCs (B) are shown using pseudocolor density plots and 5% colored contour plots for each tissue type. Each dendritic cell subset can be followed in the gating strategy: pDC (orange); pre-DC (pink); cDC1 (yellow); cDC2 (blue).