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7 protocols using recombinant human il 1ra

1

Neurotransmission Modulation Assay

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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).
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2

Intestinal Organoid Secretion Assay

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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.
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3

Quantification of Secreted Factors from MSCs

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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).
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4

Pharmacological Manipulation of Neuroinflammation

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We purchased CGP55845A, DNQX, and AP-5 from Tocris Bioscience (Ellisville, MI), recombinant mouse IL-1β from Biolegend (San Diego, CA), recombinant human IL-1ra from Peprotech (Rocky Hill, NJ), and ethanol from Remet (La Mirada, CA). Drugs were dissolved in ACSF by adding a known concentration of the stock solutions. Drug concentrations for IL-1β, IL-1ra, and ethanol were selected based on our previous reports33 (link),53 (link).
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5

Molecular Mechanisms of Inflammation

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Constructs: HNF4α siRNA (Sigma-aldrich), HNF4α shRNA constructed in pLV3 vector (GenePharma), Bay 11-7082 (Sigma-aldrichInc), MNU(Sigma-aldrich), Recombinant Human IL-1β(Peprotech), Recombinant Human IL-1RA(Peprotech);
Antibodies: HNF4α(CST), p65(Abcam), IL-1R1(Santa Cruz), p-p65(Cell Signaling), cagA(Abcam), ki67(Abcam)CCND1(Cell Signaling), Bcl-2(Santa Cruz), b-actin(Sigma aldrich).
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6

Quantification of Inflammatory Cytokines

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The levels of VEGF, G-CSF, IL-6, TNF-α, and IL-10 were quantified by ELISA using mouse (m) or human (h) DuoSet® kits (R&D Systems, UK) according to the manufacturer’s instructions. 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). For each assay, protein levels were calculated against a four-parameter logistic (4-PL) curve fit. Quantification limits were ~30 pg/ml for all the cytokines measured (except TNF-α, ~60 pg/ml), and no statistical analyses were carried out for values below this limit.
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7

Flow Cytometry Antibody Reagents

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PCR and qPCR primers were purchased from IDT (Coralville, IA). Antibodies specific for CD45 (552950, clone 104), CD11b (557657, clone M1/70), Ly6G (551460, clone 1A8), Ly6C (561237, clone AL-21), CD11c (550261, clone HL3) and all isotypes were purchased from BD Pharmingen (Mountain View, CA, USA). The antibody specific for F4/80 (25-4801-82, clone BM8) and its isotype were purchased from eBioscience (San Diego, CA, USA). Recombinant human and mouse IL-1α and recombinant human IL-1Ra were purchased from Peprotech (Rocky Hill, NJ, USA). The doxycycline-inducible pQCXIX RT3GEPIR construct was kindly provided by Dr. S. Olejniczak (RPCI) [9 (link)].
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