Multiparametric Immune Cell Analysis

Phenotypes of myeloid and T cells were assessed by flow cytometry as previously reported [30 (link),34 (link)]. Briefly, CD14⁺ and CD33⁺ moDCs were stained with anti-CD14 (REA599), CD33 (REA775), CD80 (REA661), CD83 (REA714), CD86 (REA968), HLA-DR (REA805) and respective isotype (IT) antibodies. CD14-depleted PBMCs were stained with anti-CD3 (REA613), CD4 (REA623) and CD8 (REA734) antibodies. Dead cells were excluded by propidium iodide or DAPI staining. EVs were bound to 3.9 µm beads (Invitrogen, Thermo Fisher Scientific, Waltham, MA USA) and stained with anti-CD63 (REA1055), CD81 (REA513), or respective IT as reported previously [14 (link)]. All antibodies and dyes were purchased from Miltenyi Biotec, Bergisch Gladbach, Germany. EVs and cells were acquired on FACS Calibur (BD Biosciences, Heidelberg, Germany) and MACSQuant^®Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany). Data were analyzed by FlowJo V10.7.2. (BD Biosciences, Heidelberg, Germany).