Luminex magpix instrument

Cytokine levels were determined using a multiplexed bead immunoassay and measured with a Luminex MAGPIX instrument (Luminex, Austin, TX, USA) as previously detailed [22 (link), 23 (link)]. In brief, TNF‐α and CXCL10 were measured using magnetic bead assay (Mouse XL Cytokine Luminex Performance Premixed Kit; Bio‐Techne, Minneapolis, MN, USA) following manufacturer's instructions. Each assay was analyzed on the Luminex MAGPIX instrument to measure inflammatory concentrations followed by a 5‐parameter logistic curve‐fitting method from a standard curve of each respective analyte. Plasma samples were normalized by equivalent volume of commercially supplied dilution buffer and examined in duplicate per each assay run. Concentration was calculated by the statlia Immunoassay Analysis software (Brendan Bioanalytics, Carlsbad, CA, USA).

The freshly drawn blood samples were stimulated ex vivo in whole blood cultures via lipopolysaccharides (LPS). Therefore, samples were immediately diluted 1:5 with the cell culture medium RPMI 1640 including 20 mmol HEPES and L-glutamine (Sigma-Aldrich, Hamburg, Germany) and added antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin; Sigma-Aldrich, Hamburg, Germany). The samples were seeded into 12-well microtiter plates and mixed with 10 ng/mL (final concentration) LPS from Escherichia coli (Sigma-Aldrich, Hamburg, Germany). The plates were incubated for 24 h at 37 °C without a CO₂ application. The used HEPES buffer is able to stabilize the pH over 24 h. The supernatants were frozen at −80 °C until analysis.
The levels of the inflammatory markers (IL-1β, IL-6, IL-10, TNF-α, MCP-1, MMP-9) in whole blood culture supernatant were simultaneously determined using a human Magnetic Luminex Assay (Bio-Techne, Abingdon, Oxon, UK) and a Magpix Luminex instrument (Luminex Corp, Austin, TX, USA).

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), IFN-γ -inducible protein (IP)-10, interleukin (IL)-1RA, IL-6, and tumor necrosis factor (TNF)-α were measured using a human cytokine / chemokine magnetic bead panel (Milliplex MAP kit, Merck Millipore, Billerica, MA, USA), a Magpix Luminex instrument and Xponent software (version 4.2 Luminex Corp, Austin, Texas, USA) according to manufacturer's instructions. Standard curves using a 5-parameter logistic regression were applied to calculate concentrations of cytokines. Antigen- and mitogen-induced cytokine production was calculated by subtraction of non-stimulated background concentrations from sample concentrations. Measurements below the limit of quantification were set to 0.1 pg/ml, measurements above the limit of quantification were set to 10,000 pg/ml (calibration range: 3.2–10,000 pg/ml). A valid positive control was defined as an uncorrected cytokine concentration > 20 pg/ml (26 (link)). A valid negative control was defined as uncorrected cytokine concentration < 20 pg/ml. In cases where the nil concentration was higher than 20 pg/ml, the positive control had to be higher than the nil concentration.