Milliplex map kit

IL-10, IL-17A, IFN-γ and IL-13 were measured in the culture supernatants using multiplex immunoassays (Millipore, Milliplex Map Kit). IL-21 (eBioscience), IL-4 (eBioscience, High Sensitivity) and IL-6 (Peprotech) were detected using ELISA in accordance with the manufacturer’s protocol. IFN-γ, IL-10, IL-12p40, IL-13 and IP-10 (CXCL10) levels were determined in patients’ plasma samples (Millipore, Milliplex Map Kit). The levels of the cytokines were quantified by reference to standard curves. As the standards were not covering all values obtained from the samples, median values of the readings in “Luminex” and optic density measurements in ELISA were used for the statistical analysis. Results were expressed as pg/ml and ng/ml in the figures. In the PBMC cultures, the induced levels for each cytokine were calculated by subtracting the cytokine levels in the supernatants of non-stimulated wells from those that were CD3-stimulated.

All blood sampling was completed by a certified phlebotomist. Approximately 10 mL of blood was collected via venipuncture from the antecubital vein into vacutainer tubes containing ethylene diamine tetra-acetic acid (EDTA); tubes were immediately inverted multiple times to prevent coagulation. Blood was centrifuged at 1500×g for 15 min at 4 °C and the resultant plasma was aliquoted into microtubes and stored at − 80 °C until analysis. Myokine analysis was performed in duplicate with a Luminex MAGPIX flow cytometer (Luminex Corp., Toronto, ON, Canada). IL-4, IL-6 and IL-7 were analyzed with a Human High Sensitivity T-Cell Magnetic Bead Panel assay (Milliplex Map Kit, EMD Millipore Corp, Billerica, MA, USA). LIF and irisin were quantified with a Human Myokine Magnetic Bead Panel assay (Milliplex Map Kit, EMD Millipore Corp). Plasma nitrate/nitrite (NO_X) was determined in duplicate using an enzyme-linked immunosorbent assay (ELISA; Nitrate/Nitrite Colorimetric Assay Kit, Cayman Chemical, Ann Arbor, MI, USA). All assays were completed according to the manufacturer’s recommendations. The intra-assay coefficient of variation for the ELISA ranged from 3.1% to 7.1% while the inter-assay coefficient was 5.93%. The coefficient of variation for the multiplex assays ranged from 7.2% to 23.8% while the inter-assay coefficient of variations ranged from 6.77% to 18.8%.

The plasma levels of interferon-inducible protein-10 (IP10)(also known as CXCL10), monocyte chemoattractant protein-1 (MCP-1) (also known as CCL2), and macrophage inflammatory protein-1β (MIP-1β) (also known as CCL4) were measured using a Millipore cytokine three-plex panel assay (MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel)(Milliplex MAP kits, EMD Millipore, Billerica, MA, USA). The plasma levels of interferon gamma (IFNγ), tumor necrosis factor-α (TNF-α), interleukin-1β(IL-1β), IL-6, IL-8 (also known as CXCL8), and IL-17A were determined using a Millipore cytokine seven-plex panel assay (MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel) (Milliplex MAP kits, EMD Millipore, Billerica, MA, USA). All analyses were performed by T.Y. Chen according to the manufacturer’s protocol. The data were read using a Luminex 200 system (Luminex, Austin, TX, USA). Values of these cytokines and chemokines were reported as pg/ml. Data on cytokines and chemokines were collected and analyzed using an instrument equipped with MILLIPLEX Analyst software (EMD Millipore). For these nine cytokines/chemokines, the intra-assay laboratory coefficients of variation were less than 8% and the inter-assay coefficients of variation were less than 10%.

The plasma levels of chemokine CXCL10 also known as interferon-inducible protein-10 (IP10), chemokine CCL2 also known as monocyte chemoattractant protein-1 (MCP-1), and chemokine CCL4 also known as macrophage inflammatory protein-1beta (MIP-1β) were measured using a Millipore cytokine three-plex panel assay (MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel)(Milliplex MAP kits, EMD Millipore, Billerica, MA, USA). The plasma levels of interferon gamma (IFNγ), tumor necrosis factor-alpha (TNF-α), interleukin-beta (IL-β), IL6, chemokine CXCL8 also known as IL8, and IL17A were determined using a Millipore cytokine seven-plex panel assay (MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel) (Milliplex MAP kits, EMD Millipore, Billerica, MA, USA). All analyses were performed by T.Y. Chen according to the manufacturer’s protocol. The results were read using a Luminex 200 system (Luminex, Austin, TX, USA). Values of these cytokines and chemokines were reported as pg/ml. Data on cytokines and chemokines were collected and analyzed using an instrument equipped with MILLIPLEX Analyst software (EMD Millipore). For these 9 cytokines, the intra-assay laboratory coefficients of variation were less than 8% and the inter-assay coefficients of variation were less than 10%.

Serum was collected from sacrificed mice (unless otherwise noted) at various time points following infection with C. albicans in WT and Nos3^-/- mice. A Luminex bead array Milliplex MAP Kit (catalog no MPXMCYTO-70K, Millipore, Billerica, MA) was used to quantify IL-1a, IL-6, IL-10 IL-17, IL-12p40 TNF-β, MIP-1β and GMCSF. IL-9, IL-15, MIP-1α and MIP-1β Cytokines were analyzed in sera from an independent experiment, according to the manufacturer’s specifications.

Supernatants were collected 24 h after seeding of the cells and centrifuged to remove debris before assay. A human cytokine/chemokine MILLIPLEX MAP kit (Millipore) was used to measure GRO/CXCL1, IL-6, CXCL10, and IL-1β concentrations in supernatants following the manufacturer’s protocol. A Luminex 100/200 System was used to run the plate, compute standard curves, and calculate cytokine and chemokine concentrations.