Cd1c bv421

PBMCs were stained with the Zombie Aqua Flexibility Viability dye (BioLegend, San Diego, CA, USA) for the live/dead gate for 15 min at room temperature, followed by the antibodies for (1) TLR stimulation experiments: CD3-PE-Cy7, CD19-APC-H7, CD56-BV421, CD16-BUV395, HLA-DR-BB515, CD11b-BV605, CD11c-APC (all from Becton Dickinson, Franklin Lakes, NJ, USA) and CD14-PEDazzle594 (BioLegend); and (2) time-course experiments: a lineage-negative (lin⁻) cocktail (CD3/CD19/CD20/CD56/CD14/CD16-AF700, Bio-Rad, Hercules, CA, USA), CD14-PEDazzle594, HLA-DR⁺-BB515 (Biolegend), CD123-PE (BioLegend), CD11b-BV605 (Becton Dickinson), CD11c-APC (Becton Dickinson), CD141-BV711 (Becton Dickinson), CD1c-BV421 (BioLegend) and CD1aPerCP/Cy5.5 (Biolegend) for 20 min on ice, washed twice and resuspended in flow cytometry buffer (phosphate-buffered saline (PBS) + 2% fetal bovine serum (FBS)).
All samples were run on an LSR Fortessa X-20 (Becton Dickinson) with a minimum of 100,000 events recorded per sample. All data were analyzed with FlowJo Software v10.7.1 (Ashland, OR, USA).

For analysis of surface marker expression by flow cytometry, cells were harvested in FACS buffer (1% BSA, 2mM EDTA) and transferred to a 96-well v-bottom plate. For surface marker staining, all antibodies were mixed in PBS and 30 µL staining mix was added per well. Staining was performed for 30 min at 4 °C in the dark. Thereafter, cells were washed once and fixed in 4% PFA for 15 min at room temperature following two washing steps with FACS buffer. A FACS Canto II flow cytometer (BD Biosciences, San Francisco, CA, USA) was used to assess median fluorescence intensities (MFI) of single, living CD1c⁺ primary DCs using the following antibodies: CD1c-BV421 (L161, Biolegend, Biozym, Vienna, Austria), Fixable Viability Dye-eFluor506 (eBioscience, Thermo Fisher Scientific, Vienna, Austria), CD40-FITC (5C3, eBioscience), CD86-PE (IT2.2, eBioscience), HLA-DR-APC (LN3, eBioscience), CD14-PerCP-Cy5.5 (MɸP9, BD), PD-L1-PE-Cy7 (MIH1, BD). Data analysis was performed on FlowJo 10 Software.