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6 protocols using recombinant mouse il 1β

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

Neuroinflammation and α-Synuclein Pathology

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LPS (Escherichia coil O111:B4) used for cell culture studies was purchased from Calbiochem (San Diego, CA; cat# 437627) and for animal studies was purchased from Sigma-Aldrich (St. Louis, Mo; cat# L3012). Anti-tyrosine hydroxylase (TH) and anti-Iba-1 were purchased form EMD Millipore (Burlington, MA) and Wako (Richmond, VA), respectively. The secondary antibodies were purchased from Vector Laboratories (Burlingame, CA). The rat anti-mouse CD-11b antibody was purchased from abD Serotec (Raleigh, NC, cat# MCA711G). Anti-pro-IL-1β, anti-alpha-synuclein (α-synuclein), and anti-3-Nitrotyrosine (3-NT) antibodies were purchased from Abcam (Cambridge, MA). Mouse interleukin-1 receptor antagonist (IL-1Ra), NLRP3 inhibitor MCC950, caspase-1 inhibitor Z-YVAD, IRE1α (inositol-requiring enzyme 1α) inhibitor 4μ8C, TNF-α, and IL-1β ELISA kits were purchased from R&D Systems (Minneapolis, MN). Tauroursodeoxycholic acid (TUDC) was from Selleckchem (Houston, TX). Mouse IL-1β pro-form ELISA kit was from eBioScience (San Diego, California). Recombinant mouse IL-1β was from BioLegend (San Diego, CA). Cell culture ingredients were obtained from Invitrogen (San Diego, CA). All other reagents came from Sigma Chemical Co. (St. Louis, MO).
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3

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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4

Flow Cytometry and ELISA Immunoassays

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Fluorochrome-conjugated monoclonal antibodies (mAbs), including PE-CD11b (M1/70, 101207, 1:400), PerCP/Cy5.5-IL-17A (TC11-18H10.1, 506919, 1:100), PE/Cy5-Gr-1 (RB6-8C5, 108409, 1:600), APC-CD4 (GK1.5, 100412, 1:400), FITC-TNF-α (MP6-XT22, 506304, 1:100), and APC/Fire750-CD4 (GK1.5, 100460, 1:100) were purchased from BioLegend (San Diego, CA), while PE-IL-17A (eBio17B7, 12-7177-81, 1:100) was purchased from eBioscience (San Diego, CA). Anti-mouse IL-17A neutralizing monoclonal antibody (17F3, BP0173) was purchased from BioXCell (West Lebanon, NH). Recombinant mouse IL-1β, IL-6, IL-17A, and TNF-α were purchased from BioLegend. Mouse IL-17A ELISA kit was purchased from BioLegend. Mouse IL-17A ELISPOT kit was purchased from R & D Systems (Minneapolis, MN). Native chicken and bovine collagen type II were purchased from Sigma-Aldrich (St. Louis, MO) and Chondrex (Redmond, WA), respectively.
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5

MyD88-Targeting Compound Synthesis

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We purchased CGP 55845A, DNQX, and DL-AP5 from Tocris Biosciences (Ellisville, MI, USA) and recombinant mouse IL-1β from BioLegend (San Diego, CA, USA). We obtained ethanol from Remet (La Mirada, CA, USA). Hydrocinnamoyl-L-valyl-pyrrolidine (AS-1)—a low-molecular-weight MyD88 mimetic—was synthesized as previously described [42 (link)].
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6

Isolation and Culture of Murine Abdominal Stromal Cells

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Primary cultures of abdominal stromal cells were prepared from peritoneal wall biopsies and mesentery of adult mice. The tissues were washed in PBS and digested in 0.25% trypsin-EDTA solution for 50 min at 37°C with gentle agitation. The mixture was filtered on a 70-µm cell strainer and centrifuged at 1,500 rpm for 5 min. Cells were cultured in DMEM supplemented with 15% heat-inactivated FBS, penicillin (100 U/ml), streptomycin (100 µg/ml) and amphotericin B (250 ng/ml) until confluence. Cells were subcultured in DMEM containing 10% FBS and used at passage 3. For experimentation, 1.5×105 cells were seeded in 6-well plates, starved for 24 h and incubated for 3 h with 100 µg/ml PTX, 10 ng/ml recombinant mouse IL-1β (BioLegend) or PBS as control.
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