200 system

Multianalyte profiling was performed to measure inflammatory markers secreted in cell culture medium from hGFs at 6, 24, and 48 h after induced inflammation using a Luminex 200 system. Median fluorescent intensity was analyzed using a 5‐parameter logistic line‐curve fitting method for calculating analyte concentrations in samples via the xPONENT 3.1 software (Luminex). Levels of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), monocyte chemoattractant protein‐1 (MCP‐1), and tumor necrosis factor alpha (TNF‐α) were measured using Human Cytokine/Chemokine Magnetic Bead Panel kit. The assay was performed according to the protocol provided by the manufacturer.
Total and phosphorylated (pS536) NF‐κB p65 levels in hGFs were determined 0.5 h after induced inflammation using the respective Human InstantOne™ ELISA kits (Invitrogen) according to the protocol provided by the manufacturer.

Expression of inflammatory cytokines was measured using the Bio-rad Bio-Plex Pro Human Cytokine 27-Plex Assay kit, read on a Luminex 200 system and analyzed using Luminex xPotent Software as per product specifications. The evaluated cytokines consisted of: basic fibroblast growth factor (bFGF), eotaxin, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFNg), interleukin-1 receptor antagonist (IL-1RA), Interleukins (IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17a, interferon gamma inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP-1a, MIP-1b), platelet derived growth factor bb (PDGF-bb), regulated on activation normal T-cell expressed and secreted, tumor necrosis factor alpha (TNFa), vascular endothelial growth factor (VEGF). Plasma samples were measured at baseline (pre-treatment), from either non-surgically managed (n=44) or surgical (n=39) patients, or post-surgically at 6-weeks (n=31), 3 months (n=24), 6 months (n=22) or 1 year (n=17). Samples from the different groups and time-points were randomly allocated to plates, and the experiment was conducted using de-identified samples run in duplicate. Cytokine concentration was calculated based on standard curve as per product manual.

Adipokines and peptides were measured as previously described (18 (link)) in all SP and BS samples collected and were measured in triplicate using a magnetic bead-based multiplex assay (Bio-Plex Pro™ Human Diabetes Assay, and Bio-Plex Pro™ Human Hormone Assay, Bio-Rad Laboratories, Inc., USA) according to the manufacturer’s protocol. Data acquisition was performed by a Bio-Plex 200 System™ which uses Luminex fluorescent-bead-based technology (Luminex) to facilitate the analysis of up to 100 different families of color-coded polystyrene beads and allow multiple measurements of the sample ensuing in the effective quantification of adipokines. The Multiplatform luminex assay offers the advantages to use low amount of serum (12.5 to 25 ul of serum per multiplex assay), decreased quality control variability by performing simultaneous assays on a single sample, improved correlated data for multiple analytes, increased sample and finally reduce the manual error during the assay. The working range of the assay is 3,500–280,266 pg/ml with a sensitivity of 0.8 pg/ml and intra-assay CV of 3–4%. Dosage was performed at the Unit of Endocrinology at the University of Rome “Foro Italico”.