Anti nkp46 bv421

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.

Fresh peripheral blood was drawn from HBV-infected and healthy children, and the red blood cells were lysed using NH₄Cl lysis solution. After washing by fluorescence-activated cell sorter (FACS) buffer, leukocytes were stained with specific fluorochrome-conjugated monoclonal antibodies (mAbs) for flow cytometric analysis. For NK cell phenotypic analysis, leukocytes were stained with mAbs, including anti-CD3-PerCP5.5, anti-CD56-APC, anti-NKp30-PE, anti-NKp46-BV421, and anti-HLA-DR-FITC (BD Biosciences, USA) mAbs. For T-cell phenotypic analysis, leukocytes were stained with mAbs, including anti-CD3-PerCP5.5, anti-CD8-APC, anti-CD4-FITC, anti-CD27-PE, anti-CD45RA-BV421 (BD Pharmingen, USA), and anti-CD38-PE-Cy 7 (Biolegend, USA) mAbs. Intracellular IFN-γ-BV421 (BD Biosciences, USA) and INF-α-PE-Cy 7 (Biolegend, USA) were detected after PMA/lonomycin mixture (250×, BioLegend, USA) for 1 h and Monensin Solution (1,000×, EB eBioscience, USA) at 37°C for 4 h. Multiparameter flow cytometry was performed using a CytoFLEX flow cytometer (Beckman Coulter, USA), and data were analyzed using FlowJo software V10.