Bio plex 200 array reader

We simultaneously analyzed a selection of 14 cytokines, namely BDNF, intercellular cell adhesion molecule-1 (ICAM-1), interleukin (IL)-10, IL-8, IL-6, inducible protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), platelet-derived growth factor BB (PDGF-BB), tumor necrosis factor α (TNF-α) and β (TNF-β), vascular cell adhesion molecule-1 (VCAM-1), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor 1 (VEGF-R1) and 2 (VEGF-R2). Cytokine concentrations were quantified in duplicate using a magnetic bead 14-plex panel assay (Human ProcartaPlex Mix&Match 14-plex, PPX-14-MX9HJ62; Thermo Fisher Scientific, Waltham, USA) performed according to the manufacturer’s recommended protocol and read using a Bio-Plex 200 array reader (Bio-Rad, Hercules, California, USA). The standard curve was based on five-parameter nonlinear regression. Each cytokine concentration was then calculated by the curve.

Multiplex bead-based immunoassays on a Bio-Plex® 200 Array Reader (Bio-Rad Laboratories, Munich, Germany) were used to measure HGF, EGF, TGF-β1, IL-6, IL-8, Apo2, PLGF and VEGF. For details see Appendix.

Cytokines were analyzed with thawed supernatants using human Bio-Plex Pro^™ Human Cytokine 27-plex, Th17 Cytokine Panel or custom arrays with a Bio-plex 200 array reader (Bio-Rad, Hercules, CA). For human confirmation of THP1 results, some LTA1 treated samples were above the interpolatable range for MIP-1β and IL-8 respectively. These values were replaced with 0.01 over the highest interpolated value for that cytokine.

Heparinized plasma glucose, triglycerides, total and HDL-cholesterol concentrations in samples were determined using enzymatic methods and spectrophotometry (Modular 700, Roche Diagnostics, Meylan France). Low-density lipoprotein (LDL)-cholesterol was calculated by Fried-wald’s formula [Total cholesterol–(HDL cholesterol + triglycerides/5)]. Serum insulin was measured by immunoradiometric sandwich assay (Bis-Insulin IRMA^®, CisBio international, Gif-Sur-Yvette, France). The homeostasis model assessment resistance (HOMA-RI) index was calculated by the following equation: insulin (μIU/mL) x glucose (mmol/L)/22.5. Serum high-sensitive C-reactive protein (hsCRP) level was measured using automated immunonephelometry (Behring Nephelometer II Analyzer^®, Dade Behring, Germany). Leptin and TNFα were measured by commercially available multiplex beads immunoassays (Fluorokine MAP Multiplex Human Cytokine Panel and Obesity Panel, R&D Systems, Minneapolis, USA) and read by the Bioplex 200 array reader (Bio-Rad Laboratories, Hercules, CA, U.S.A.) which uses Luminex xMAPTM Technology (Luminex Corporation, Austin, TX, U.S.A.).

A customized Luminex assay (Magnetic Luminex assay, R&D Systems) was used to measure the plasma concentration of most of the markers identified by O-link, as some were not available for testing with the Luminex technology. Assays were performed according to the supplier’s instructions using) the Luminex xMAP Technology (Luminex Corporation) and read on the Bio-Plex 200 array reader (Bio-Rad Laboratories). Five-parameter logistic regression curve (Bio-Plex Manager 6.0) was used to calculate sample concentrations. In addition to previously reported biomarkers (IL-1Ra, MCP1, IL-6, IL-10, MIP1b, and TNF-α) (24 (link)), additional markers from the O-Link analysis were MCP2, MCP3, MCP4, CXCL10, OSM, CX3CL1, MCSF, CXCL11, TRAIL, RANKL and IL15 were measured in both Geneva and US cohort. All data below thresholds (last point of the standard curve) were set to half the value of the corresponding threshold.

For NK cell activation, IL-12p70 and IL-18 were purchased from Peprotech (Rocky Hill, NJ) and MBL International (Woburn, MA), respectively. IL-15-amplified NK cells were stimulated for 18 h with 1.3 nM IL-12 and 22.2 nM IL-18 in optimized NK cell medium. As a non-activated control, a portion of NK cells were incubated with 50 nM IL-15 in optimized NK cell medium. After overnight stimulation, a small sample of supernatant was removed from both IL-12/18 and IL-15 cultures and IFN-γ levels were quantitated by xMAP cytokine bead array (EMD Millipore, Billerica, MA) using a BioPlex 200 Array Reader (Bio-Rad, Hercules, CA). NK cells were washed with 2x 50 ml portions of PBS and resuspended to a final concentration of 1–1.5x10⁷ cells/ml in PBS + 0.05% BSA. Groups of CAST/EiJ were inoculated IP with 3–3.5x10⁶ cells in 0.2 ml.
For NK cell-labeling experiments, IL-12/IL-18 activated NK cells were washed 2x with 50 ml of PBS and resuspended to a final concentration of 5x10⁶ cells/ml in 1 μM CFSE (Thermo Fisher Scientific, Waltham, MA) in PBS. After staining for 10 min in the dark at room temperature, CFSE labeling was quenched by addition of ice-cold cRPMI. Cells were washed with 50 ml of PBS and resuspended to a final concentration of 1–1.5x10⁷ cells/ml in PBS + 0.05% BSA. Groups of CAST/EiJ were inoculated IV with 2-3x10⁶ cells in 0.2 ml.