Cd16 pecy7 3g8

Single-cell suspensions were prepared from organs by cutting tissues into small fragments, followed by enzymatic digestion for 30 minutes at 37°C with Collagenase D (2 mg/mL) (Roche) and DNAase (50 μg/mL) in RPMI-1640 medium. Tissue fragments were further disrupted by passage through a 70-μm nylon cell strainer (BD Biosciences). Red blood cells were lysed with BD Pharm Lyse buffer (BD Biosciences). Cells were then stained for viability with Zombie NIR (BioLegend) for 30 minutes at room temperature, washed twice with PBS, and blocked with Human TruStain FcX (Fc receptor blocking solution, BioLegend) for 20 minutes at room temperature. Cells were stained with an antibody cocktail consisting on the following multiparametric flow cytometry panel: CD45-FITC (2D1; 368507, BioLegend), CD1c-APC (L161; 331523, BioLegend), CD38–PerCP-Cy5.5 (HIT2; 303521, BioLegend), CD16-PECy7 (3G8; 302015, BioLegend), CD141-PE (M80; 344103, BioLegend), HLADR-BV785 (L243; 307641, BioLegend), CD8-BV570 (RPA-T8; 301037, BioLegend), CD14-BV510 (M5E2; 301841, BioLegend), CD56 (HCD56; 318325, BioLegend), CD19-PB (HIB19; 982404 BioLegend), CD4-BUV737 (SK3; 564305, BD Biosciences), and CD3-BV650 (OKT3; 317323, BioLegend). Formaldehyde-inactivated samples were acquired using an LSRFortessa (BD Biosciences) and analyzed with FlowJo software.

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).