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9 protocols using tnf α

1

Synovial Fluid Cytokine and Adipokine Profiling

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The right knee joints were opened to collect synovial fluid shortly after sacrifice using the Whatman chromatography paper method52 (link). Samples were weighed, diluted and centrifuged40 (link). Serum and synovial fluid cytokines and adipokines were quantified using a Rat 27 Multiplex Discovery Assay with Luminex®xMAP technology (Eotaxin, EGF, Fractalkine, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, IL-17A, IL-18, IP-10/CXCL10, GRO/KC, IFN-γ, TNF-α, G-CSF, GM-CSF, MCP-1, leptin, LIX, MIP-1α, MIP-2, RANTES, VEGF; Eve Technologies, AB, Canada).
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2

Oxidative Stress and Inflammatory Biomarkers

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Serum malondialdehyde (MDA) was measured with a thiobarbituric acid reactive substances kit using fluorometric procedures (TBARS Assay Kit, Cayman Chemical Company, Ann Arbor, MI). Lipopolysaccharide-binding protein (LBP) was measured in lithium heparinized plasma diluted 1:200 with the provided assay buffer using an enzyme-linked immunosorbent assay kit (Hycult Biotech, Plymouth Meeting, PA). Plasma total antioxidant capacity (TAC) was measured using a colorimetric assay (Antioxidant Assay Kit, Cayman Chemical Company, Ann Harbor, MI). Immediately after centrifugation, 100 μL of fresh plasma was diluted 1:50 with the provided assay buffer. TAC of plasma was defined as all antioxidants within a sample that inhibited the oxidation of 2,2’-Azino-di-3-ethylbenzthiazoline sulphonate by metmyoglobin relative to a water-soluble tocopherol analog and quantified as millimolar Trolox equivalents as described by Miller et al. (1993) (link). Serum cytokines were measured using a 13-plex Immunoassay targeting IFNy, IL-10, IL-12, IL-18, IL-1α, IL-1β, IL-4, IL-8, TNF-α, GM-CSF, IL-1ra, and IL-2 (Eve Technologies, Calgary, AB). A CV threshold of less than 5% was used for all biomarker analyses.
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3

Multiplex Serum Cytokine Analysis in Fasted Rats

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Following 12 h of fasting, rats were anesthetized with isoflurane and a blood sample was collected by cardiac puncture. Blood was centrifuged at 3000 rpm for 15 min at 4 °C and serum stored in aliquots at − 80 °C until analyzed. Rats were sacrificed by heart excision. Serum cytokines and adipokines were quantified using a Rat 27 Multiplex Discovery Assay with Luminex® xMAP technology (eotaxin, EGF, fractalkine, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, IL-17A, IL-18, IP-10/CXCL10, GRO/KC, IFN-γ, TNF-α, G-CSF, GM-CSF, MCP-1, leptin, LIX, MIP-1α, MIP-2, RANTES, VEGF; Eve Technologies, Calgary, AB, Canada).
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Biomarker Profiling in Metabolic Study

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Plasma levels of insulin, adiponectin, TNF-α, MCP-1 were measured by Eve Technologies Corp., and plasma levels of AST and ALT were measured by the Biomedical and Obesity Research Core at UNL.
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5

Multiplex Cytokine Profiling of Cell Supernatants

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Cell supernatants were collected and frozen down at -80 °C until assayed using a Human 42 Multiplex Discovery Assay with Luminex xMAP technology (EGF, Eotaxin-1, FGF-2, Flt-3L, Fractalkine, G-CSF, GM-CSF, GROα, IFNα2, IFNγ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IL-18, IP-10, MCP-1, MCP-3, MDC, MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES, sCD40L, TGFα, TNFα, TNFβ, VEGF-A; Eve Technologies, AB, Canada). The level of VEGF, IL-6, and PGE2 in cell culture supernatants were also evaluated using ELISA (Human DuoSet ELISA Development Kits (VEGF, IL-6, PGE2), R&D Systems) with absorbance measured at 450 nm/570 nm.
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6

Plasma Cytokine Profiling in Mice

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Blood was collected from the submandibular vein of all experimental mice into K2EDTA-coated tubes (BD Biosciences, 365974) and immediately placed on ice. The blood was centrifuged for 15 minutes at 900g and 4°C, and plasma was collected. Aliquots of plasma were diluted twofold in PBS and stored at –80 °C. Blood plasma cytokines were analysed through a commercially available multiplex panel including Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, CXCL1, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1alpha, MIP-1beta, MIP-2, RANTES, TNF-α, and VEGF (Eve Technologies, Alberta, Canada, MD-31).
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7

Western Blotting and Cytokine Profiling

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Cells were lysed in RIPA buffer containing Complete Mini EDTA-free protease inhibitor (Roche) and PhosSTOP phosphatase inhibitor (Roche, Mississauga, ON, Canada). Total protein was quantified and equal concentrations were submitted to SDS-PAGE by standard methods. Proteins were transferred to PVDF membrane and probed with antibodies raised against GFAP (Sigma, St. Louis, MO, USA), GS (Abcam, Cambridge, MA, USA), phospho-p38 and pan-p38 MAPK (Cell Signaling, Danvers, MA, USA), IκBα (Santa Cruz, Dallas, TX, USA), TNF-α (R&D Systems, Minneapolis, MN, USA), and GAPDH (Calbiochem, San Diego, CA, USA), and detected with appropriate IRDye secondary antibody (Li-Cor Biosciences, Lincoln, NE, USA). Blots were imaged with an Odyssey infrared imaging system (Li-Cor Biosciences). For TNF-α and multiplex cytokine profiling, conditioned media was snap frozen and submitted for laser bead analyses on a Bioplex 200 to detect sensitive and quantitative target protein concentrations against a standard curve (Eve Technologies).
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8

Gastric Cytokine Profiling in Mice

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Gastric tissue from male mice was homogenized in liquid nitrogen with a disposable pestle (Sigma-Aldrich, St. Louis, MO). One hundred fifty microliters of lysis buffer, 500 µL of RIPA buffer with protease inhibitor (Thermo Fisher Scientific, Waltham, MA), 5-µL protease inhibitor, and 5-µL 0.5 M EDTA were added. Samples were placed in a rotating mixer at 4°C for 1 hour. Supernatant was collected following centrifugation at 10,000 × g for 10 minutes at 4°C. Protein concentration was measured using a BCA kit (Thermo Fisher Scientific, Waltham, MA) and adjusted to 1 mg/mL. Thirty-two-plex gastric tissue cytokine array was performed to quantify eotaxin, G-CSF, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17A, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNFα, and VEGF-A (Eve Technologies, Calgary, Alberta, Canada).
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9

Comprehensive Body Composition Analysis

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One day prior to sacrifice, animals underwent a dual x-ray absorptiometry (DXA) scan (Hologic ODR 4500; Hologic Inc., Marlborough, MA, USA) under light anesthetic (isoflurane). Lean mass (g), fat mass (g), body fat %, bone mineral content (g), and bone mineral density (BMD) (g/cm2) were assessed using Hologic QDR software for small animals. Following 12 h of feed deprivation, rats were anesthetized with isoflurane and blood collected from the portal vein. From this sample, a Milliplex Rat Cytokine Array/Chemokine Array (Millipore, St. Charles, MO, USA) was used to measure serum TNFα, IL-1α, IL-1β, IL-5, IL-10, IL-18, and leptin (Eve Technologies, Calgary, AB, Canada). Rats were subsequently killed by overanesthetization and decapitation. The cecum, colon, and jejunum were excised, cleaned, weighed, snap-frozen, and stored at −80 °C.
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