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Dendritic cells have been investigated for cell-based immunotherapy for various applications. The low abundance of dendritic cells in blood hampers their clinical application, resulting in the use of monocyte-derived dendritic cells as an alternative cell type. Limited knowledge is available regarding blood-circulating human dendritic cells, which can be divided into three subsets: type 2 conventional dendritic cells, type 1 conventional dendritic cells, and plasmacytoid dendritic cells. These subsets exhibit unique and desirable features for dendritic cell-based therapies. To enable efficient and reliable human research on dendritic cell subsets, we developed an efficient isolation protocol for the three human dendritic cell subsets, resulting in pure populations. The sequential steps include peripheral blood mononuclear cell isolation, magnetic-microbead lineage depletion (CD14, CD56, CD3, and CD19), and individual magnetic-microbead isolation of the three human dendritic cell subsets., Competing Interests: Competing interestsMiltenyi Biotec GmbH was as part of the public-private DC4Balance consortium. All reagents purchased to perform these experiments and the MultiMACS were bought by Radboud University Medical Center., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.)