Recombinant human il 6

HASCs (10 × 10³ cells/cm²) were seeded into 24‐wells plates and cultured in αMEM containing 1% PSF, 10 IU/ml heparin, and 2% human PL in a humidified atmosphere containing 20% O₂ (normoxia [standard condition]) or 1% O₂ (hypoxia), and allowed to attach for 24 hr at 37°C. The next day, the medium was replaced with osteogenic medium, consisting of αMEM with 1% PSF, 10 IU/ml heparin, 2% human PL, 50 µM ascorbic acid‐2‐phosphate (vitamin C; Sigma‐Aldrich), 5 mM β‐glycerophosphate (Sigma‐Aldrich) and 10 nM 1,25‐(OH)₂vitamin D₃ (Sigma‐Aldrich). Recombinant human IL‐4 (R&D Systems, Minneapolis, MN), and/or recombinant human IL‐6 (R&D Systems) and recombinant human IL‐6Rα (R&D Systems) were added to the osteogenic medium. IL‐4 or IL‐6 were added in a final concentration of 1 and 10 ng/ml, respectively, and IL‐4 in combination with IL‐6 in a final concentration of 10 ng/ml. After addition of osteogenic medium supplemented with the cytokines, hASCs were incubated in 1% O₂ or 20% O₂ at 37°C during 3 days. Then, the medium was replaced by osteogenic medium without cytokines, and refreshed every 3 days during 11 days. hASCs were harvested after 2, 7, and 14 days of culture for analysis of proliferation, osteogenic differentiation, and VEGF expression as described below.

The level of phosphorylation was determined in cell lysates collected from one million vector control and shMUC2-2.2 cells that were treated with recombinant human IL-6 (25 ng/mL) (R&D Systems, Minneapolis, MN, USA) for 30 min. Relative phosphorylation levels were determined using Human Phospho-RTK Arrays (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Cell lysates were used immediately or stored at −80 °C and were incubated with a human phosphorylation array panel antibody cocktail at RT for 1 h. After blocking, the membranes were incubated with cell lysates at 4 °C overnight on a rocking platform. The membranes were subsequently washed and incubated with a freshly diluted detection antibody cocktail for 2 h at RT on a rocking platform. The membranes were subsequently washed and incubated with diluted streptavidin conjugated to horseradish peroxidase (HRP) for 30 min at RT on a rocking platform. The membranes were developed with Chemi Reagent Mix and exposed to X-ray film. The signal intensity of the array was scanned and quantified by densitometry using an AlphaImager 2200 system (Alpha Innotech) and normalized to the positive control.

Cryopreserved PBMCs were thawed and stained with viability dye (eFluor 780; eBioscience). Antibodies against CD4 (FITC or V500), CD25 (APC), CD45RA (Alexa Fluor 700)(BD Biosciences), CD127 (PE-eFluor 610), FOXP3 (PE), TIGIT (PerCP-eFluor710) (eBioscience), Helios (Pacific Blue; Biolegend). Purified anti-FCRL3 antibody was provided by Nagata (37 (link)), and was detected with F(ab′)2 anti-mouse IgG (PE-Cy7; eBioscience). Flow cytometry analysis was performed on an LSR Fortessa analyzer (BD Biosciences), and sorting throughout this study was performed on a FACS Fusion cell sorter (BD Biosciences). Recombinant human IL-6, IL-27, CLC, IL-11 (R&D systems), and LIF (Peprotech) were added to suppression assays where indicated at the time of activation.