Cd14 pe cf594

After Zombie™ dye and Fc‐block (both BioLegend, San Diego, CA, USA) pre‐incubation, PBMC were stained with anti‐human CD14‐FITC, CD19‐PerCP‐Cy5.5, CD40‐PE‐Dazzle, CD69‐FITC, CD86‐BV421, CD95‐PE (all BD Bioscience, NJ, USA), CD4‐PE‐Cy7, CD8‐PE, CD14‐PE‐CF594, CD19‐FITC, CD20‐APC‐Cy7, CD24‐PerCp‐Cy5.5, CD25‐BV605, CD27‐PacificBlue, CD38‐FITC, CD80‐PE‐Cy7 and major histocompatibility complex (MHC)‐II‐APC (all BioLegend, CA, USA). Cytokines were evaluated after adding 1‐µL Golgi‐Plug (BioLegend) to CpG‐stimulated cells for 4 h; 500 ng/mL ionomycin and 20 ng/mL phorbol 12‐myristate 13‐acetate (PMA; Sigma Aldrich, MO) were added after 2 h. Cells were stained with anti‐human interleukin‐ (IL‐)6‐FITC, tumor necrosis factor (TNF)‐A700 and IL‐10‐PE‐CF594 (all BioLegend).

Venous blood was drawn into an ethylenediaminetetraacetic acid (EDTA) S-monovette (Sarstedt, Nümbrecht, Germany) for immunophenotyping. Whole blood (100 µl per flow cytometry panel) was directly stained in FACS tubes with fluorescence-labeled antibodies (BD-Biosciences, Franklin Lakes, United States: CD8-PerCP-Cy5.5, CD5-PE, CD45RA-PE-CF594, CD19-PE-Cy5, CD4-PE-Cy7, CD45RO-APC, CD38-A700, CD3-APC-H7, CD138-BV421, CD10-BV510, CD27-BV605, CD14-PE-CF594, CD19-PE-Cy5, CD25-PE-Cy7, CD16-A700, CD3-APC-H7, CD11b-BV421, HLADR-BV510; Biolegend, San Diego, United States: CD11c-BV605; Miltenyi Biotec, Bergisch-Gladbach, Germany: BDCA1-APC, BDCA2-PE) for 15 min in the dark at room temperature (RT). After washing, red blood cell (RBC) lysis was performed with 2 ml ACK lysing buffer (Thermo Scientific, Waltham, United States) for 7 min in the dark at RT. 2 ml of FACS buffer (phosphate buffered saline, 3% fetal calf serum, 1% sodium azide) was added to wash the samples twice. Subsequently, cells were resuspended in 400 µl FACS-buffer. After the staining procedure, cells were measured by flow cytometry (LSR Fortessa cytometer, BD Biosciences, Franklin Lakes, United States) and analyzed with FACS-Diva Software (BD Biosciences, Franklin Lakes, United States). The gating strategy is available in the supplementary data section of this paper (Supplementary Figure S7).

Prior to antibody incubation, cells were stained with viability dye (Zombie™ dye, 1:500, Biolegend) for live cell/dead cell discrimination and incubated with Fc receptor blocking solution (Human TruStain FcX, BioLegend, CA) to prevent unspecific antibody binding. Extracellular antigens were stained using anti-human cluster of differentiation (CD)4-PE-Cy7, CD8-PE, CD14-PE-CF594 and CD19-FITC/PerCP-Cy5.5, CD20-APC-Cy7, CD25-BV605, CD27-PacificBlue, CD38-FITC, CD80-PE-Cy7, CD150-BV-421, major histocompatibility complex class II (MHC-II)-APC (all Biolegend, CA), CD19-PerCp-Cy5.5, CD40-PE-Dazzle, CD69-FITC, CD86-BV421, and CD95-PE (all BD Biosciences, NJ) antibodies. For analysis of intracellular cytokines, cells were permeabilized by adding fixation/permeabilization solution (Cytofix/Cytoperm, BD Biosciences, NJ) and stained with anti-human interleukin (IL)-6-FITC, IL-10-PE/CF594, and tumor necrosis factor (TNF)-Alexa Flour 700 (all BD Biosciences, NJ) antibodies. Apoptosis was evaluated using propidium iodide-PE and annexin V-FITC (both BioLegend, CA). Samples were analyzed using a LSRII Fortessa; FACS Diva (BD Biosciences) and FlowJo software were used to quantify flow cytometric data.