Anti cd16 buv395

Cell surface staining was performed on NK cells after 18h of incubation with Control-CM, ALG-CM, or 3mM CaCl₂ in R10 complete medium. Cells were stained in staining buffer with fluorochrome-linked monoclonal antibodies: anti-CD14-FITC (BD Biosciences), anti-CD3-FITC (Biolegend), anti-CD56-AF700 (BD Biosciences), anti-CD16-BUV395 (BD Biosciences), anti-CD69-BUV737 (BD Biosciences), anti-DNAM-1-PE-Vio770 (Miltenyi), anti-NKp30- anti-BV421 (BD Biosciences), anti-NKp46-BV786 (BD Biosciences), anti-NKG2D-BV650 (BD Biosciences), anti-KIR2D-PE (Miltenyi), anti-KLRG1-APC-Vio770 (Miltenyi), anti-CD95-APC (Biolegend), anti-CD85j-PE-Cy5 (BD Biosciences), anti-CD158e1/e2 PerCP-Vio700 (Miltenyi), anti-TIGIT-BV605 (Biolegend), and Fixable Viability Dye eFluor 506. The expression of activating or inhibitory receptors was analyzed on NK cell subsets based on CD16 and/or CD56 expression. Samples were analyzed using an LSRFortessa cytometer (BD Biosciences), and results were generated by FlowJo software.

All cells were incubated with FcR blocking reagent (130-059-901; Miltenyi Biotec) for 10 min at 4°C before cell surface staining. For NK cell phenotyping, PBMCs and IHLs were stained with a LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (L34966; Invitrogen) and anti-CD3/CD14/CD19-APC-Cy7 (Biolegend), anti-CD56-BUV737 (BD Biosciences), anti-CD16-BUV395 (BD Biosciences), anti-NKp46-BV421 (BD Biosciences), and anti-ILT2-PE-Cy7 (Biolegend) antibodies for 30 min at 4°C. For ILT2 ligand analysis, hepatocytes from HCC tissue (Ca) and adjacent non-cancerous liver tissue (NCa) were incubated with PE-HLA-G and isotype control. All samples were acquired on an LSRFortessa (BD Biosciences) with FACSDiva software and analyzed using FlowJo 10.8.1 software (BD Biosciences). The antibodies used for the flow cytometry analysis are listed in Supplementary Table 3.

UltraComp eBeads^TM Compensation Beads (Thermofisher) were used to optimize fluorescence compensation settings for multi-color flow cytometric analysis at a Symphony flow cytometer. UltraComp eBeads^TM were stained with the following fluorochrome-labeled anti-human antibodies: anti-CD8–BUV805 (1:200, clone SK1), anti-CD4–BUV496 (1:50, clone SK3), anti-CD86–BUV737 (1:50, clone 2331 FUN-1), anti-CD141–BUV615-P (1:50, clone 1A4), anti-CD56–BUV563 (1:50, clone NCAM 16.2), anti-CD16–BUV395 (1:50, clone 3G8), anti-CD123–BB660-P (1:50, clone 7G3), anti-CD80–BB630 (1:50,clone L307.4), anti-CD21–BV785 (1:50, clone B-ly4), anti-CD27–BV750-P (1:40,clone L128), anti-BAFF-R–BV650 (1:50, clone 11C1), anti-CD94–BV605 (1:50, clone HP-3D9), anti-CD40–APC-R700 (1:50, clone 5C3) (all BD bioscience); anti-CD3–PerCP-Vio700 (1:50, clone REA613) (Miltenyi Biotec); anti-CD57–FITC (1:100, clone TB01), anti-CD14–PE-Cy5.5 (1:200, clone TuK4), fixable viability dye eFluor780 (1:1000) (all eBioscience); anti-CD24–BV711 (1:50, clone ML5), anti-CD19–BV510 (1:25, clone HIB19), anti-HLA-DR–BV570 (1:40, clone L243), anti-IgM–BV421 (1:100, clone MHM-88), anti-CD11c–APC (1:40, clone 3.9), anti-CD38–PE/Dazzle 594 (1:100, clone HB-7), anti-CD10–PE-Cy5 (1:50, clone HI10a), and anti-IgD–PE-Cy7 (1:100, clone IA6-2) (all BioLegend).