Monocyte purification kit

NCI-N87, HCC1954, and SKBR3 cells were obtained from the Shanghai Cell Line Bank. RPMI1640 medium (61870036), Freestyle 293 medium, Dynabeads CD4 Positive Isolation Kit (11331D), TMB substrate, and fetal calf serum (FCS) were obtained from Thermo Fisher Scientific. PE-labeled goat anti-human IgG (409304), streptavidin-PE (405203), anti-human-CD3 (clone HIT3a), and anti-human-CD28 (clone CD28.2) were obtained from Biolegend. Trastuzumab and humanized anti-PD-L1 scFV were prepared in house. Cell Counting Kit-8 (CCK8) was obtained from Dojindo Laboratories. Human antibody germline sequence and primers were synthesized by Sangon Biotech. IL-4 and GM-CSF were obtained from R&D Systems, and all other recombinant proteins were products of Sino Biological. SYPRO Orange was obtained from Sigma-Aldrich. Human IFN-γ ELISA Kit (EHC102g) was obtained from Neobioscience. Cytotoxicity detection kit (LDH) was from Roche Life Science. Matrigel was obtained from BD Biosciences. Polyethylenimine (PEI) was obtained from Polysciences. Protein A probes were obtained from Pall Corporation. Protein A-Sepharose Column was obtained from BioVision. HRP-conjugated goat anti-human IgG (H + L) was obtained from Jackson ImmunoResearch. Monocyte purification kit was obtained from Miltenyi Biotec. Ficoll gradient was obtained from GE healthcare. Human PBMCs were obtained from Allcells.

Dendritic cells (DCs) were generated by culturing monocytes isolated from PBMCs using a monocyte purification kit (Miltenyi Biotec) in vitro for 7 days with 500-units/mL interleukin-4 and 250-units/mL GM-CSF (R&D Systems). CD4⁺ T cells (1 × 10⁵ cells) and allogeneic DCs (1 × 10⁴ cells) were co-cultured with different concentrations of antibodies. After 5 days, interferon gamma (IFN-γ) secretion in culture supernatants was analyzed using ELISA (R&D Biosciences).