Apc human cd3 antibody

SMMC-7721 cells were infected with different adenoviruses for 96 h, following which the culture supernatants were collected to detect the binding of αCD3HAC to PD-L1 on 7721-PD-L1 cells by flow cytometry. For direct-binding assay, 1 × 10⁵ cells were suspended in 100 μL of the collected supernatant and mixed with 5 μL of allophycocyanin (APC)-His tag antibody (#362605, Biolegend, USA). The cells without any supernatant were used as negative control, whereas the cells with APC-human PD-L1 antibody (#374514, Biolegend, USA) were used as positive control. After culturing at room temperature for 30 min, the cells were detected using flow cytometry (FACS). For the competition assay, 5 µL of the APC-human PD-L1 antibody was added to the 7721-PD-L1 suspension of the collected supernatant. The cells with APC-isotype control (#982108, Biolegend, USA) were used as negative control, whereas the cells with only APC-human PD-L1 antibody were used as positive control. FACS was performed after culturing. The binding to CD3 was manipulated as earlier except for replacing the APC-human PD-L1 antibody with the APC-human CD3 antibody (#300312, Biolegend, USA).

The culture supernatants of SMMC-7721 cells infected with different adenoviruses were still used in the following experiments. 7721-PD-L1 cells were seeded into 96-well plates (1 × 10⁴ cells/well). The next day, PBMCs pretreated with IL-2 for 72 h were added at the effector: target (E:T) ratios of 5:1. Then, 100 uL of the supernatant was added simultaneously. The specific cytotoxicity toward 7721-PD-L1 cells was measured by the lactate dehydrogenase assay 16 h later using a CytoTox 96 nonradioactive cytotoxicity kit (#G1780, Promega, USA) following the manufacturer’s protocols. Then, the cells and the culture supernatant of every well were separated. The cells were stained with the APC-human CD3 antibody (#300312, Biolegend, USA) and the phycoerythrin (PE)-human CD69 antibody (#310906, Biolegend, USA) for 30 min at room temperature and measured using FACS to detect activated T cells. The levels of interleukin-2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in the supernatants were measured using the corresponding ELISA kits (D2050, DIF50C, and DTA00D, R&D, USA).

7721-PD-L1 and Jurkat cells were co-cultured in the supernatants collected previously at the ratio of 10:1 for 24 h. Then, the cells were separated and stained with the APC-human CD3 antibody (#300312, Biolegend, USA) and fluorescein isothiocyanate (FITC) – Annexin V (#556547, BD, USA). The apoptosis of Jurkat cells was measured using FACS.