Cd15 bv650 w6d3

The WB MDSC 9-color, 11-parameter flow cytometric assay included the following fluorescently-labeled antibodies: CD45-V500-C (2D1), CD19-FITC (HIB19), CD20-FITC (L27), CD56-PECY7 (NCAM 16.2), HLA-DR-APC-H7 (L243), CD33-PE (WM53), CD11b-APC (ICRF44), and CD14-BV421 (MΦP9) (all, BD Biosciences); CD3-FITC (SK7), CD16-BV785 (3G8), and CD15-BV650 (W6D3) (all Biolegend). Samples were treated with BD FACS™ Lysing Solution (BD Biosciences) to lyse RBC. Total MDSC were defined as CD45 + CD3⁻CD19⁻CD20⁻CD56⁻CD16⁻HLA-DR⁻CD33 + CD11b + cells, while the monocytic (M-MDSC) and granulocytic or polymorphonuclear (PMN-MDSC) subsets were defined as CD14+ and CD15+, respectively. Absolute cell numbers were obtained using Trucount tubes (BD Biosciences). Presence of intracellular Ki67 was analyzed using antibodies to CD14 (MΦP9, exclusion marker), CD56 (NCAM16.2), CD4 (SK3), CD8 (SK1) (all, BD Biosciences); CD3 (SK7) and Ki67 (both, Biolegend) as well as Fixable Viability Dye eFluor 780 and FoxP3/Transcription Factor Staining Buffer Set (both eBioscience, San Diego, CA). All samples were fixed with 2% paraformaldehyde and data acquired on the same day. Flow cytometric data analysis was performed using a BD LSRFortessa and FlowJo software v9.9.5 (Treestar, Ashland, OR). Statistical analyses utilized paired or unpaired t tests, as appropriate.

Red blood cell-lysed whole blood or cells isolated from mucosal tissues were stained using a combination of multicoloured flow cytometry panels designed to determine Fc-receptor expression on CD14+ monocytic cells, mDC or NK cells. Briefly, for overall cellular phenotyping; CD3 V450 [UCHT1], CD4 PECy7 [SK3] (Biolegend), CD8 Pacific Orange [3B5] (Invitrogen), CD19 BV650 [SJ25C1]. For CD14 and mDC FcR Phenotyping; CD3 V450 [UCHT1], CD14 Qdot 605 [TüK4] (Invitrogen), CD16 Pacific Orange [3G8] (Invitrogen), CD11c A700 [B-ly6], CD123 PECy5 [9F5], CD32 APC [FLI8.26], CD64 APC H7 [10.1], CD89 PE [A59], CD19 FITC [HIB19]. For NK cell FcR phenotyping; CD15 BV650 [W6D3] (Biolegend), CD16 PECy7 [3G8] (Biolegend), CD66b FITC [G10F5] (Biolegend), CD64 APC H7 [10.1], CD56 PECy5 [HCD56] (Biolegend), CD45 A700 [HI30] (Biolegend), CD89 PE [A59], CD3 V450 [UCHT1], CD32 APC [FLI8.26]. Unless otherwise specified, all antibodies were sourced from BD Biosciences. Anti-FcRs were able to detect antibody-occupied FcR as indicated by the manufacture and in house controls. Dead cells were excluded from analysis through staining with Aqua Viability Dye (Invitrogen).