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

## TL;DR

This paper provides a detailed protocol for isolating and analyzing dendritic cell subsets from blood and lymphoid organs of non-human primates and humans using flow cytometry.

## Contribution

The paper introduces a comprehensive and optimized protocol for ex vivo phenotyping and purification of dendritic cell subsets using flow cytometry.

## Key findings

- Steps for isolating mononuclear cells from blood, lymph nodes, and spleen are detailed.
- Optimized antibody panels are provided for identifying DC subsets via flow cytometry.
- Protocols for cytokine production analysis and high-purity sorting of DC subsets are described.

## 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

•Steps for isolating a single-cell suspension from blood, lymph nodes, and spleen of NHP•Guidance on identifying DC subsets via flow cytometry with optimized antibody panels•Steps for ex vivo stimulation and analysis of cytokine production by DC subsets•Protocol for high-purity sorting of DC subsets for downstream assays

Steps for isolating a single-cell suspension from blood, lymph nodes, and spleen of NHP

Guidance on identifying DC subsets via flow cytometry with optimized antibody panels

Steps for ex vivo stimulation and analysis of cytokine production by DC subsets

Protocol for high-purity sorting of DC subsets for downstream assays

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

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.