Pai 1

Sandwich enzyme-linked immunosorbent assay (ELISA) kits were used to quantify plasma concentrations of proinflammatory cytokines (Leptin, monocyte chemotactic protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1)), angiogenic factors (vascular endothelial growth factor (VEGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1)) and insulin following manufacturers' protocols. Leptin, MCP-1, PAI-1, VEGF and TIMP-1 ELISA kits were obtained from R&D Systems (Minneapolis, MN), and the insulin kit was from Mercodia (Winston-Salem, NC). Samples were read within the linear range of the assay, and the accuracy of the analysis was confirmed by the controls provided in each kit.

The commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the levels in pleural effusions or in PMC conditioned medium of thrombin (AssayPro LLC., St. Charles, MO, USA), TNF-α, IL-1β, PAI-1, and tPA (R & D System; Minneapolis, MN, USA) as previously described [20 (link)].

Mature 3T3-L1 adipocytes were treated with various doses of aspirin and stimulated by 2.5 ng/mL TNF-α (PeproTech, Rocky Hill, NJ, USA) and 1 mg/mL lipopolysaccharide (LPS) (Sigma) in DMEM containing 1% BS for 24 h. Cell culture supernatants were collected and cytokine levels were analyzed by enzyme-linked immunosorbent assay (ELISA). The analytic protocol of cytokines included IL-6, IL-1β, macrophage chemoattractant protein-1 (MCP-1), leptin, adiponectin, plasminogen activator inhibitor-1 (PAI)-1 (R&D, Minneapolis, MN, USA), and vascular endothelial growth factor (VEGF) (PeproTech, Rocky Hill, NJ, USA), according to the manufacturer’s instructions.

Cell culture supernatants (48-hours) were analyzed in duplicate by ELISA using kits: PGE2 (multi-species), rat IL-1RA, rat IL-10 (R&D Systems, Minneapolis, MN); FGF7/KGF (BlueGene, Shanghai); MPO (RayBioTech, Norcross, GA); TSG6 (MyBiosource, San Diego, CA). Other cytokines were evaluated on a Luminex (Austin, TX) using rat-specific bead sets: FasL, G-CSF, GM-CSF, IFNγ, IL-4, IL-12p40&p70, LIX, RANTES (Millipore, Billerica, MA); PAI-1, CXCL1, TNFα, IL1α, IL-2, IL-4, IL-6 (R&D Systems).

Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to perform the biochemical measurements of adipokines and insulin according to the manufacturer’s instructions (visfatin kit was from BioVision; chemerin, PAI-1, resistin, CCL-2, IL-6, IL-8, IL-10 and TNF-α kits were from R&D; leptin, adiponectin and insulin kits were from Thermo Fisher Scientific). The coefficient of variability (CV) for the ELISA kits were shown as follows: visfatin (intra-assay: 4.4–8%; inter-assay: 8.2%), chemerin (intra-assay: 3.9%; inter-assay: 7.3%), PAI-1 (intra-assay: 6.8%; inter-assay: 7%), resistin (intra-assay: 4.7%; inter-assay: 8.4%), CCL-2 (intra-assay: 5%; inter-assay: 5.1%), IL-6 (intra-assay: 2.6%; inter-assay: 4.5%), IL-8 (intra-assay: 4.7–6.7%; inter-assay: 5.8–7.7%), IL-10 (intra-assay: 3.7%; inter-assay: 6.9%), TNF-α (intra-assay: 4.9–7.8%; inter-assay: 4.7%-5.8%), leptin (intra-assay: 3.9%; inter-assay: 5.3%), adiponectin (intra-assay: 3.8%; inter-assay: 5.5%), and insulin (intra-assay: 4.8%-6%; inter-assay: 8.1% to 9%). All measurements were performed in duplicates or triplicates. Seven controls (one blank, two at lower concentrations, two at medium concentrations, and two at higher concentrations) were also quantified in duplicates to check for the reproducibility of measurements and confirm acceptable reproducibility.