Anti tgf β antibody

PBMCs were isolated from peripheral blood using Ficoll density-gradient centrifugation. Naïve CD4⁺T cells were purified from PBMCs according to the manufacturer’s instruction (Miltenyi Biotec, Bergisch Gladbach, Germany). These purified naïve CD4⁺T cells (1 × 10⁶/well) were differentiated into Tfh cells under Tfh cell-polarizing condition (3 μg/ml soluble anti-CD3/28 (eBioscience, San Diego, CA, USA), 50 ng/ml recombinant IL-6 (rIL-6, PeproTech Inc, Rocky Hill, NJ, USA), 50 ng/ml rIL-21 (Abcam, Cambridge, MA, USA), 10 μg/ml anti-IL-4 antibody (eBioscience), 10 μg/ml anti-IFNγ antibody (eBioscience) and 10 μg/ml anti-TGF-β antibody (R&D, Minneapolis, MN, USA)) for 3 days. After initial culture, these differentiating Tfh cells were washed with PBS for 2 times and further expanded alone or cocultured with UC-MSCs (1 × 10⁵/well) in the presence of 3 μg/ml soluble anti-CD3/28 for another 2 days. To detect the factors involved in UC-MSCs-mediated suppression, UC-MSCs were collected after 2 days’ coculture with differentiating Tfh cells and then were fixed by Trizol. Furthermore, 100 μM 1-methyl-DL-tryptophan (1-MT, Sigma), the inhibitor of IDO enzyme activity or 10 μg/ml anti-IL-10 antibody (eBioscience) or 10 μg/ml anti-HLA-G antibody (Biolegend, San Diego, CA, USA) was added to the MSCs-Tfh cells coculture system to block their effects on Tfh cells.

To identify the molecular weights of proteins in the supernatant samples, SDS-PAGE was performed using ASC sup and control sup, according to the manufacturer’s instruction Bio-Rad Laboratories, Hercules, CA, USA). The electrophoresed gels were transferred to Hybond-C extra nitrocellulose membranes (Amersham Biosciences, Little Chalfont, UK) as performed in a previous study [18 (link)]. TGF-β in the ASC sup was measured using an anti-TGF-β antibody (10 μg/g body weight in 200 μL PBS; R&D Systems, Minneapolis, MN, USA).

THP-1 derived macrophages and lung cancer cells were co-cultured using a cell culture insert (Corning, NY, USA) with a porous membrane to separate the upper and lower chambers. The macrophages (7.5×10⁵ cells/well) were seeded into the upper chamber of the transwell apparatus, and the A549 and H1299 cells were placed in the lower chamber (3×10⁵ cells/well). The chambers with the THP-1-derived macrophages were then placed directly on top of six-well plates containing the A549 and H1299 cells, and the resulting co-culture systems were incubated for 24 h or 48 h in serum-free RPMI 1640. Lung cancer cells and THP-1-derived macrophages were cultured for 24 h in serum-free RPMI 1640 as controls. To study the role of TGF-β, anti-TGF-β antibody (R&D Systems, Minneapolis, MN, USA) was added to the co-culture system.