Cd16 apc cy7

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.

One milliliter of blood collected in sodium heparin tubes was stimulated with 10 ng IFNβ or unstimulated for 24 h. Proteomic Stabilizer PROT1 (Smart Tube, Inc.) was added and incubated for 10 min at room temperature prior to freezing at −80°C. Samples were thawed, and single‐cell suspensions of blood leukocytes were obtained using Thaw‐Lyse buffer (Smart Tube, Inc) according to manufacturer's instructions. Following incubation with human Fc receptor blocker, TruStain FcX (Biolegend Inc.), and Super Bright Complete Staining Buffer (Thermo Fisher), cells were stained with fluorescently conjugated antibodies: CD14 BV421, CD64 BV785, HLA‐DR FITC, Siglec‐1 PE, CD38 APC, CD19 BV650, CD66b PerCP/Cy5.5, CD16 APC/Cy7, CD86 PE/Cy7, and CD3 BV510 from Biolegend, acquired with a Cytek Northern Lights 3000 (Cytek Biosciences) and analyzed using FlowJo.

Lysosomal–associated membrane protein-1 (LAMP-1 or CD107a), a glycosylated membrane protein primarily found in lysosomes, is a marker for degranulation of NK cells (26 (link)). Accordingly, CD107a is utilized as a cytotoxicity marker for functional assays. Therefore, anti-CD107a-PE-Cy5 mAb (Biolegend, San Jose, CA, USA) was added to the co-culture of PBMCs and human erythromyeloid leukemia cell line K562, with an effector/target (E:T) ratio of 10:1 for 4 hours at 37°C in the presence of anti-CD107a-PE/Cy5 mAb (Biolegend, San Jose, CA, USA). PBMCs alone were used as unstimulated control. Following the incubation, cells were stained with anti–CD3-Alexa Fluor700, -CD16-APC/Cy7 and CD56-BV650 mAbs (all from Biolegend, San Jose, CA, USA) for 20 minutes in the dark. To evaluate perforin and granzyme B content of PBMCs, samples were fixed and permeabilized according to the manufacturer protocol (Cytofix & Cytoperm Kit, Biolegend, San Jose, CA, USA), and then stained with intracellular anti-perforin-PerCP/Cy5.5 and -granzyme B-PE (Biolegend, San Jose, CA, USA) mAbs for 20 minutes. The samples were washed with PBS and were analyzed with NovoCyte flow cytometer running NovoExpress software (Agilent Technologies, USA).

PBMCs were incubated with mixtures of fluorochrome-conjugated antibodies, and identified by flow cytometry. Data acquisition and analysis were performed using a BD CANTO II Flow Cytometer and BD FACS DIVA software (BD Biosciences, Flanklin Lakes, New Jersey). Dead cells were distinguished by 7-amino-actinomycin D (7-AAD, BD Biosciences) staining. Cells were not permeabilized for intra-staining. To identify monocyte lineage cells, the surface markers CD14-PE (Biolegend, San Diego, California, clone HCD14) and CD16-APC/Cy7 (Biolegend, clone 3G8) were used. Pro-inflammatory and anti-inflammatory phenotypes were characterized using S100A9-FITC (BioRad, Hercules, California, clone MAC387) and CD163-APC (Biolegend, clone RM3/1) antibodies (Supplementary Table). S100A9 and CD163 expression levels were determined using a positive dataset for each antibody, identified using a matched concentration of mouse IgG1 kappa isotype control for fluorochrome color (Biolegend, clone MOPC21).

The following fluorescence-labeled monoclonal antibodies (mAb) were used: CD3-FITC or PE/Cy5 (clone UCHT1), CD11c-FITC (3.9), CD16-APC/Cy7 (3G8), CD30-PE/Cy7 (BY88), CD33-PE, BV605 or PE/Cy5 (P67.6), CD56-PE/Dazzle™594 (N901), CD57-FITC (HCD57), CD62L-PE/Cy7 (DREG56), CD107-BV510 (H4A3), CD117-BV421 (104D2), KLRG1-APC/Fire 750 (SA231A2), PD1-APC/Cy7 (EH12-2H7), NKG2D-PE (1D11), NKp46-BV510 (9E2), NKp44-APC (P44-8), NKp30-BV785 (P30-15), Granzyme B-Pacific Blue (GB11), IFNγ PE/Cy7 (B27), Perforin-PE (dG9), TNFα PE/Dazzle™ 594 (all from Biolegend, CA, USA), CD3 PerCP-Vio700 (REA613), CD14-PerCP-Vio700 (REA599), CD33-APC, CD56 (REA196), anti-biotin-VioBright515 and 7-AAD (all Miltenyi Biotec), NKG2C-AF700 (134591) from R&D systems (MN, USA), and CD158b1,b2,j-PE/Cy5 (GL183), NKG2A-APC (Z199) purchased from Beckman Coulter (CA, USA). Flow cytometric analyses were performed on a CytoFLEX (Beckman Coulter) or MACSQuant 10 Analyzer (Miltenyi Biotec). Data analysis was performed on Kaluza 2.1.1 software.