Recombinant human il 1ra

We purchased CGP 55845A, DNQX, and DL-AP5 from Tocris Biosciences (Ellisville, MI, USA), recombinant mouse IL-1β from Biolegend (San Diego, CA, USA), recombinant human IL-1ra from Peprotech (Rocky Hill, NJ, USA), and TTX from Calbiochem (San Diego, CA, USA). We obtained ethanol from Remet (La Mirada, CA, USA).

Small intestinal organoid generation from mouse jejunum (section taken 8–13 cm from the gastric pylorus), maintenance, and two-dimensional (2D) culture were carried out as previously described [50 (link)]. GIP secretion experiments were performed using 2D organoid-derived cultures, 18–24 h after seeding cells onto 48-well plates. Cells were washed three times in warm saline buffer (138 mM NaCl, 4.5 mM KCl, 4.2 mM NaHCO₃, 1.2 mM NaH₂PO₄, 2.6 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES; adjusted to pH 7.4 with 1 M NaOH, and supplemented on the day of experiment with 1 mM glucose and 0.1% fatty acid-free bovine serum albumin) before incubation in saline buffer for 30 min at 37 °C. The buffer was then completely removed before test reagents, dissolved in 150 µL saline buffer, were added to the organoid cultures. The test reagents used were 100 ng/mL LPS (Sigma-Aldrich, St. Louis, MO, USA), 100 µg/mL recombinant human IL-1Ra (PeproTech, Rocky Hill, NJ, USA), 10 ng/mL recombinant human MCP-1 (Peprotech), or 10 µM forskolin plus 10 µM 3-isobutyl-1-methylxanthine (IBMX) plus 10 mM glucose (all Sigma-Aldrich, St. Louis, MO, USA). After incubation for 2 h at 37 °C, supernatants were removed and centrifuged at 2000× g for 5 min at 4 °C, transferred to a fresh tube, and snap-frozen on dry ice.

Levels of human IL-10, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), TNF-α and G-CSF in culture media from MSCs were quantified by ELISA using DuoSet® kits (R&D Systems, UK) according to the manufacturer’s instructions. Human IL-1Ra levels were measured using an ELISA kit from Peprotech (UK) combined with external standards prepared using recombinant human IL-1Ra (National Institute for Biological Standards and Controls (NIBSC), UK). Quantification limits in human ELISAs were 10 pg/ml for IL-1Ra, 15 pg/ml for G-CSF, NGF, TNF-α and VEFG, and 25 pg/ml for BDNF and IL-10. ELISA kits for mouse IL-6, TNF-α, IL-10 and G-CSF (all quantification limits ~30 pg/ml) were purchased from R&D Systems and used following the manufacturer’s instructions. For each assay, samples were diluted as needed and protein levels were calculated against a four-parameter logistic (4-PL) curve fit. All values are expressed as mean ± standard error of the mean (SEM).