Anti cd137 mab

For CD137 detection, the PBMCs or TILs of GC patients were placed in flow tubes, and 5 µL each of an anti-CD45-PerCP antibody (BioLegend, USA), anti-CD3-FITC antibody (BioLegend, USA) and anti-CD137-APC antibody (BioLegend, USA) was added. The cells were incubated in the dark for 10 min and washed with PBS once. PBS (200 µL) was added for flow cytometry detection.
For examination of CD8⁺ T cells proliferation, CD8⁺ T cells of GC patients were placed in flow tubes and washed once with PBS. PBS (200 µL) was added for flow cytometry detection.
For NF-κB detection, CD8⁺ T cells from GC patients were treated with 10 µg/ml anti-CD137 mAb (BPS Bioscience, USA) for 72 h and placed in flow tubes. After washing once with PBS, a Fixation/Permeabilization Solution (BD Cytofix/Cytoperm™) was added at room temperature for 30 min. After washing once with PBS, an NF-κB p65 rabbit mAb (1:1000, CST, USA) was added. The cells were incubated in the dark for 1 h and washed once with PBS. Five microliters of anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor^® 594 Conjugate) (1:500, CST, USA) was added, and the cells were incubated in the dark for 30 min. After washing once with PBS, 200 µL of PBS was added for flow cytometry detection.

PBMCs (1 × 10⁵) and primary GC cells (2 × 10⁴, CFSE stained) were mixed, placed in anti-human CD3 antibody-coated 96-well plates containing 200 µL of 10% FBS (Hyclone, USA) DMEM, and treated with 10 µg/ml anti-CD137 mAb (BPS Bioscience, USA). Apoptosis in the GC cells was detected using flow cytometry after 72 h.