OMIP‐44: 28‐Color Immunophenotyping of the Human Dendritic Cell Compartment

Update / Correction. In the original version of this article, plasmacytoid DCs (pDCs) were gated as CD45+ live CD14− CD3− CD19− CD56− CD123+ cells. This gating scheme does not exclude basophils, which also express CD123 and may be present in mononuclear cell preparations, particularly Ficoll/Percoll-based isolation protocols, despite their polymorphonuclear nature. The pDC population shown in the original Figure 1 included such a contaminating basophil population. We updated the gating scheme to gate on CD45+ Lin− HLA-DR+ cells before identifying CD123+ pDCs (see updated Fig. 1), which effectively eliminates basophils from the pDC gate. This changes the mean pDC frequency in Online Figure 5 from 4.81% to 3.07%, while in Online Figure 6 the legend should read “total CD123+ cells”. The updated gating tree does not affect any other aspects of the OMIP. Overview gating of an example 30-parameter dataset on cryopreserved PBMCs. Cryopreserved PBMCs were obtained from AllCells, stained and subsequently aquired on a BD FACSymphony. Optimal PMT voltages were determined by voltage titration experiments and iterative refinement. All reagents were titrated prior to use. For Figures (C) till (G), files were concatenated from two separate aquisitions. Additional gating and staining controls are shown in Online Figure 3–5. (A) Gating strategy for live CD45+ singlets, after exclusion of debris and doublets. (B) Overview of the 28 markers analyzed within this experiment. (C) Gating strategy for major immune cell lineages (CD14+ classical monocytes, CD19+ B cells and CD3+ T cells). (D) CCR7 and CD45RA expression on CD4+ and CD8+ T cells. (E) After exclusion of CD14+ CD19+ CD3+ events, gating strategy for CD56+ NK cells and lineage-negative HLA-DR+ cells, followed by identification of CD123+ plasmacytoid DCs and canonical subsets within conventional CD11c+ HLA-DR+ DCs. Color shading indicates the populations used for histogram overlays. (F) FMO control for CD80 expression on CD19+ B cells. (G) Differential expression of selected markers on the indicated DC subsets: CD123+ pDCs (green), double negative DCs (orange), CD141+ cDC1 (blue) and CD1c+ cDC2 (red) as gated in (E). (H) Overview of selected markers on CD11c+ HLA-DR+ DCs including the corresponding fluorescent-minus-one (FMO) controls (lower panel). BUV: Brilliant Ultraviolet, BV: Brilliant Violet, BB: Brilliant Blue, AF: AlexaFluor, PE: Phycoerythrin, APC: Allophycocyanin. We also want to clarify that the “DN DC” population purposefully included CD16+ cells. Given the technical nature of an OMIP our focus was to describe an optimized panel for identifying different myeloid cell surface markers, while remaining agnostic regarding the biological heterogeneity. This “DN DC” population includes both non-classical monocytes and some DCs that might share phenotypic and functional properties (see original references #8 and #13).