Cd45 po

Leucocytes subsets were identified using an antibody panel containing CD45‐PO (Life Technologies) for lymphocytes; CD123‐PerCPCy5.5 (BD Pharmingen), CD203c APC (Sony), HLA‐DR‐PB (Sony), and CD41‐PE‐Cy7 for basophils; and CD45‐PO (Life Technologies) and CD14‐APC‐H7 (BD Pharmingen) for monocytes. To distinguish between the three different subsets of dendritic cells (DCs), an antibody panel containing HLA‐DR‐PE‐CY7 (BioLegend), CD11c‐PB (BioLegend), and CD123 PerCP Cy5.5 (BD Pharmingen) was used for plasmacytoid dendritic cells (pDCs). For two subsets of myeloid dendritic cells (mDCs), CD14‐V500 (BD), HLA‐DR‐PE‐CY7 (BioLegend), CD1c‐APC‐Cy7 (BioLegend), and CD141‐APC (Miltenyi) were used. Leucocyte subset quantities were depicted as the percentage of cells within the total leucocyte measures/numbers.

Staining D, the percentages of classical monocytes, intermediate monocytes, and non-classical monocytes were determined using a 5-color membrane staining. Thawed PBMC used for staining C (see above) were also used for further monocyte subset analysis. A total of 1 x 10⁶ PBMCs were stained with a cocktail of CD45-PO (Life Technologies), CD64-APC (BD Pharmingen), CD66b-BV421 (BD Horizon), CD14-APC-H7 (BD Biosciences), CD16-PE-Cy7 (Invitrogen) for 30 minutes at RT, lysed with BD Lysing solution (BD Biosciences) for 10 min at RT and washed with PBS pH 7.8. Monocytes were measured using a BD FACS Lyric flowcytometer. The analysis was performed using BD FACSSuite software. Monocytes (CD45⁺CD64⁺CD66^-) were defined as: classical monocytes (CD14⁺⁺CD16^-), intermediate monocytes (CD14⁺⁺CD16⁺), and non-classical monocytes (CD14⁺CD16⁺⁺) (49 (link)). For the monoclonal antibodies used in staining D, see Supplementary Table S1.

Leukocyte subsets were identified using an antibody panel containing CD45-PO (Life Technologies) for lymphocytes, CD3-AF 700 (BioLegend) for T cells, CD14-PeCy7 (BioLegend) for monocytes, CD19-BV711 (BioLegend) for B cells, and CD16/CD56-APC (eBioscience, BD Biosciences) for NK cells. T cell subsets were distinguished by CD3-AF 700 (BioLegend), CD4-BV711 (BioLegend), CD8-PeCy7 (BD), CD27-APC-eF780 (eBioscience, San Diego, CA, USA), and CD45RO-PB (BioLegend). Each sample was also stained with isotype-matched control monoclonal antibodies for spectral compensation and to correct for background fluorescence. Because of the extensive antibody panel, some antibodies were measured in the same fluorescence channel, e.g., CD4 and CD19, CD8 and CD14, and CD16 and CD56. A representative example of the gating strategy to identify the different lymphocyte subsets is provided in Figure S1 in Supplementary Material.