Anti cd107a h4a3

Cells from cultures for 0, 5, 10, and 15 days were labeled using following PE-, FITC-, APC-, or PerCP-conjugated mouse antihuman antibodies in fluorescence activated cell sorter buffer (BD Biosciences, USA) for 30 min at room temperature; the antibodies included anti-CD16 (3G8), anti-NKp30 (P30-15), anti-NKG2D (1D11), anti-DNAM-1 (11A8), and anti-CD107a (H4A3) (Biolegend, USA) as well as CD3-FITC and CD56-PE (BD Biosciences). APC- and FITC-conjugated and purified antimouse IgG1 (Biolegend) were used as isotype controls. Goat antihuman IgG (Thermo Fisher Scientific) and the matched isotype control antibody were used to stain cells and to identify cells binding with Herceptin and IgG. Cells were analyzed using FACS Calibur and FACS CantoII instruments (BD Biosciences, USA) and FlowJo software to determine the percentage of positive cells in CD3⁻CD56⁺ NK cell populations.

The following antibodies were used in this study: anti-B7-H3 (FM276, Miltenyi Biotech), anti-GD2 (14.G2a, BD Biosciences), human Ig (polyclonal, Thermo Fisher Scientific), anti-mouse IgG (polyclonal, R&D), anti-CD3 (UCHT1, BioLegend), anti-HisTag (J095G45, BioLegend), anti-CD34 (QBEnd10, R&D), anti-ab-TCR (IP26, BioLegend), anti-CD107a (H4A3, BioLegend), anti-cD25 (BC96, BioLegend), anti-CD69 (FN50, BioLegend), anti-Tim3 (F38-2E2, BioLegend), anti-Lag3 (11C3C65, BioLegend), anti-PD-1 (EH12.1, BD Biosciences), anti-mouse CD45 (30-F11, BioLegend), anti-human CD45 (HI30, BioLegend), Ghost Red 780 (Tonbo Biosciences), Zombie Yellow Viability Dye (BioLegend), propidium iodide (Gibco), Cell Trace Violet (Thermo Fisher Scientific), and Precision Count Beads (BioLegend).

For surface staining, cells were washed once with FACS buffer (PBS containing 0.1% BSA and 0.02% sodium azide) and cells were incubated for 15 min with the antibody mixture in FACS buffer. Cell surface staining was performed using the following antibodies: anti-CD3 (UCHT1, eBioscience™), anti-CD4 (RPA-T4, BioLegend, San Diego, USA), anti-CD8 (RPA-T8, BioLegend), anti-CD56 (HCD56, BioLegend), anti-HLA-DR (L243, BioLegend), anti-CD107a (H4A3, BioLegend), and anti-CD253/Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) (RIK-2, BD). The Fixable Viability Dye (FVD) eFluor™ 780 (eBioscience™) was used to exclude dead cells from the analysis. Cells were washed with FACS buffer and fixated with 4% PFA for up to 2 h. Cells were washed twice with Intracellular Staining Perm Wash Buffer (BioLegend) and incubated for 20 min with anti-GzmB (GB11, BD) and anti-IFNγ (XNG1.2, BD) in Intracellular Staining Perm Wash Buffer. Cells were washed again twice with Intracellular Staining Perm Wash Buffer, collected in FACS staining buffer, and stored at 4°C until acquisition. Samples were acquired with a BD LSR II flow cytometer with a HTS module and data were analyzed using FACSDiva and FlowJo Version 10.8 (both BD Becton Dickinson, Heidelberg, Germany).