V450 mouse anti human cd3

Staining of cells and analysis on a flow cytometer (FACS canto II; BD Biosciences) were done as described in our lab (59 (link)). V450 Mouse Anti-Human CD3 (Catalog: 560366, Clone: UCHT1), APC Mouse Anti-Human CD56 (Catalog: 555518, Clone: B159), PE-Cy7 Mouse Anti-Human CD16 (Catalog: 557744, Clone: 3G8), and PE Mouse Anti-Human IFN-γ (Catalog: 554701, Clone: B27) were obtained from BD Biosciences. In brief, PBMCs or NK cells were resuspended in staining buffer (0.5% BSA, 0.05% sodium azide in PBS) and pre-incubated with FcR blocking reagent (Miltenyi Biotec) for 15 min at 4°C. The cells were then simultaneously stained with suitable antibodies and then washed with staining buffer. For intracellular IFN-γ staining, cells were first fixed/permeabilized and subsequent IFN-γ staining performed according to the manufacturer’s protocol (Cytofix Buffer and Permeabilization Buffer III; Catalog:555028, BD Biosciences). The data acquired were analyzed with FlowJo Version 10 (Treestar software, Ashland, OR, USA).

1 × 10⁵ tumor cells were incubated with CFSE-labeled PBMCs (E:T = 10:1) in medium supplemented with TfR-BiTE at the concentrations indicated for 48 h. Thereafter, the cell mixture was cultured in the medium without TfR-BiTE for an additional 72 h. Then cells were stained with a mixture of V450 mouse anti-human CD3, APC-Cy7 mouse anti-human CD4, and V500 mouse anti-human CD8 (BD Biosciences). After washing, the cells were stained with 7-AAD for 15 min. Proliferation on 7-ADD⁻/CD3⁺/CD4⁺ and 7-ADD⁻/CD3⁺/CD8⁺ T-cell subsets was determined by flow cytometry.