Cd66b apc

Assays of Ab-dependent phagocytosis by monocytes (ADCP) and neutrophils (ADNP) were performed essentially as described (47 (link)–49 (link)). Briefly, 1 µm yellow-green fluorescent microspheres (Thermo, F8813) were covalently conjugated with recombinant RBD and incubated for 3 h with dilute serum or nasal wash specimens and either the human monocytic THP-1 cell line (ATCC, TIB-202), or with freshly-isolated primary neutrophils. After pelleting, washing, and fixing, phagocytic scores were quantified as the product of the percentage of cells that phagocytosed one or more fluorescent beads and the median fluorescent intensity of this population as measured by flow cytometry with a MACSQuant Analyzer (Miltenyi Biotec). ADCP assays were performed in duplicate with high correspondence between results presented here and the replicate run. ADNP assays were performed in biological replicate using neutrophils purified from two different healthy donors for which results were averaged. A subset of neutrophils was stained with CD66b-APC (BioLegend G10F5) and PI (Biotium 41007) to determine the purity and viability of the isolated cellular fraction. CR3022 and VRC01 were used as positive and negative controls, respectively. Wells containing no Ab were used to define the level of Ab-independent phagocytosis.

Transwell plates (HTS Transwell^®-96 Permeable Support with 5.0 µm Pore Polycarbonate Membrane, Corning, Corning, NY, USA) were used. Supernatants of long-term treated NK cells were collected 24 h after medium exchange. Then, 235 µL of the supernatants was added to the lower chamber and 0.25 × 10⁶ of freshly isolated PBMCs and granulocytes in 80 µL were added onto the insert. After 5 h of incubation (37 °C, 5% CO₂), plates were incubated for 10 min at 4 °C, performed to induce detachment of monocytes. Cells were then removed from the lower chamber, transferred to a v-well plate, and pelleted by centrifugation. In the next step, cells were resuspended in 50 µL FACS-buffer containing an antibody mix of CD3 PerCP (1:100; Clone:SK7, BioLegend), CD56 BV421 (1:50; Clone:NCAM16.2; BD Biosciences), CD14 FITC (1:50 Clone: 61D3; Santa Cruz Biotechnology, Dallas, TX, USA), CD19 AF700 (1:50; Clone:HIB19; BioLegend), CD66b APC (1:200; Clone:G10F5; BioLegend) and CD11c PE (1:100; Clone: B-ly6; BD BioSciences) transferred to TruCount tubes (BD BioSciences) and incubated for 20 min at RT in the dark. Measurement of cells was performed using the BD LSRFortessa. Data were analyzed using FlowJo software (FlowJo) and absolute cell counts were calculated according to the manufacturer’s instructions.

Flow cytometry was performed from organ single-cell suspensions or purified neutrophils, live cells were identified using zombie UV dye (#423108 BioLegend) or live/dead blue (#L34961 Invitrogen), intracellular cytokine staining was performed following 4 h restimulation with phorbol myristate acetate (PMA; 50 ng/mL) and ionomycin (500 ng/mL), followed by a 1h Brefeldin A (5 µg/ml) incubation. Antibodies: (all from BioLegend unless stated otherwise): CD11b FITC (1:200, M1/70), CD11b PerCP (1:100 M1/70), CD11b Bv510 (1:100, M1/70), Ly6C PerCP/Cy5.5 (1:400, HK 1-4), Ly6C PE (1:800, HK 1-4), Ly6G APC/Cy7 (1:400, 1A8), Ly6G PE (1:400, 1A8), Ly6G Bv785 (1:100, 1 A8), PD-L1 PE (1:100, 10F.9G2), CD54 AF647 (1:200, YN1/1.74), CD117 BUV395 (1:100, 2B8, BD), TNF-α BV421 (1:200, MP6-XT22), CCL2 PE (1:50, 2H5), CXCL1 AF647 (1:50, 1174A, R&D). Human neutrophils were analyzed in peripheral blood or after isolation using the MACSxpress^® kit (Miltenyi Biotech). Isolated cells were stimulated with PMA (10 ng/mL), LPS (0.1 µl/mL) and CL097 (1µg/mL) for 4 hours. Antibodies: CD16-APC-Cy7 (1:200, 3G8) and CD66B-APC (1:400, G10F5, Biolegend), viperin (RSAD2) PE (1:200, MaP,VIP; Biosience). Flow cytometry analysis was performed on a Cytek Aurora (Cytek)- or BD LSRII cytometer and data analysis were performed using FlowJo V10.4.2 software.