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5 protocols using tnf α 210 ta 020

1

AMPK Signaling in RPE Cells

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RtEGM Retinal Pigment Epithelial Cell Growth Medium (RtEGM BulletKit #195409) was purchased from Lonza (Walkersville, MD) and fetal bovine serum (#10438034) were purchased from Thermo Fisher Scientific (Waltham, MA). CFB antibody (sc-271636) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for AMPK a1 (Abcam) : 32047,AMPKa2 (Abcam) : 3760, were from Abcam  and (P-ACC (#3661), ACC (#3676), AMPK (#2603), P-AMPK (# 2535), and GAPDH (#2118)  were purchased from Cell Signaling Technologies (Beverly, MA). AICAR (#A611700), a pharmacological activator of AMPK, was purchased from Toronto Research Chemicals (Toronto, ON, Canada). 5-iodotubericidin (IODO #I100), dipyridamole (DPY #D9766) and nicotinamide (N3376) were purchased from Sigma (St. Louis, MO). TNF-α  (#210-TA-020) was purchased from R & D Systems (Minneapolis, MN).
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2

Investigating Vaspin's Modulation of Inflammatory Responses in Human Aortic Endothelial and Monocytic Cells

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Human aortic endothelial cells (HAECs) were obtained from Lonza Inc. (CC-2535, Walkersville, MD, USA) and maintained in endothelial basal medium (CC-3162, Lonza) supplemented with various growth factors that are required for the growth of endothelial cells and 2% fetal bovine serum at 37°C in a humidified incubator supplemented with 5% CO2.
THP-1 cells, a human monocytic cell line (ATCC® TIB-202™, Rockville, MD, USA), were grown in RPMI-1640 medium containing 10% fetal bovine serum. In all experiments, cells were used at six or fewer passages.
TNFα (210-TA-020, R&D Systems, Inc. Minneapolis, MN, USA) was used as the representative proinflammatory cytokine and phosphate buffered saline was used as the vehicle. Cells were transferred to medium containing 1% fetal bovine serum and incubated in media containing 10 ng/mL TNFα for the indicated amount of time. Vaspin was obtained from Adipogen Inc. (AG-40A0064, Incheon, Korea) and phosphate buffered saline was used as the vehicle. Cells were transferred to medium containing 1% fetal bovine serum and incubated in media containing various concentrations of Vaspin for the indicated amount of time before treatment with TNFα. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (123040, Calbiochem, Darmstadt, Germany), an activator of AMPK, was used as a positive control.
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3

TNF-α and CHX-Induced Cell Death

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Target cells were plated in 12-well plates at 1.2 × 105 cells per well. Twenty-four hours after seeding, cells were treated with 75 ng ml−1 TNF-α (210-TA-020, R&D Systems) and 10 μg ml−1 CHX (C1988, Sigma) for 10 h. The percentage of survival was determined based on quantification of remaining adherent cells using PrestoBlue reagent (A13262, Life Technologies Ltd.) relative to control DMSO treated cells.
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4

Cell Culture and Cytokine Treatments

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SUM149 and SUM190 cells were maintained in Ham’s F-12 w/L-glutamine (Fisher Scientific) containing 0.5 μg/mL fungizone, 5 μg/mL gentamicin, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Additionally, SUM149 cells were supplemented with 5% fetal bovine serum (FBS), 5 μg/mL insulin, and 1 μg/mL hydrocortisone (Sigma-Aldrich). SUM190 cells were supplemented with 0.1% bovine serum albumin, 5 μg/mL insulin, and 1 μg/mL hydrocortisone (Sigma-Aldrich). U937 and MDA-MB-231 cells were cultured in RPMI containing 10% FBS, 0.5 μg/mL fungizone, 5 μg/mL gentamicin, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). MCF10A cells were maintained in 50:50 DMEM:F12 media supplemented with 5% horse serum, 10 μg/mL insulin, 0.5 μg/mL hydrocortisone, 0.02 μg/mL epidermal growth factor, and 0.1 μg/mL cholera toxin (Sigma-Aldrich). SUM149 and SUM190 cells were maintained at 37 °C with 10% CO2 and all other cell lines at 37 °C with 5% CO2. Fresh 0.25% trypsin-EDTA in phosphate buffered saline (PBS) was used to re-suspend cells. Cytokines were purchased from R&D Systems and used at the following concentrations in all experiments: 600 ng/mL IL-8 (208-IL-050), 200 ng/mL TNFα (210-TA-020), 50 ng/mL CCL5 (278-RN-010/CF), 20 ng/mL IL-6 (206-IL-010), 10 ng/mL VEGF (293-VE-010), 5 ng/mL CCL2 (279-MC-010/CF), and 1 ng/mL IL-10 (217-IL-005).
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5

Activation of S1P Signaling Pathways

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LPS (L3024; Sigma-Aldrich, St. Louis, MO), TNF-α (210-TA-020, R&D, Minneapolis, MN), and IL-1β (201-LB-005, R&D) were obtained commercially. S1P was purchased from Tocris (1370; Minneapolis, MN). VPC24191, a S1PR1/S1PR3 agonist and CYM5520, a selective agonist of S1PR2, were obtained from Sigma-Aldrich. The AKT activator SC79 was purchased from Tocris.
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