Milliplex map high sensitivity human cytokine magnetic bead panel

A panel of 51 eicosanoids and lipid metabolites were measured in plasma samples using a 6490 Triple Quadrupole mass spectrometer (Agilent, New Castle, DE, USA). The individual eicosanoids were identified using metabolite-specific fragmentation and retention times¹² . We used the Milliplex MAP High Sensitivity Human Cytokine Magnetic Bead Panel (EMD Millipore Corp., St. Charles, MO) to measure four cytokines: IL-1β, IL-6, TNF-α, and IL-10. An additional inflammation marker, CRP, was also measured using a DuoSet enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN)^{10 (link)}.
We measured three protein oxidation markers in plasma samples: 3-nitrotyrosine [NY], 3-chlorotyrosine [CY], and o,o’-dityrosine [DY]. To quantify these biomarkers, total plasma protein was first precipitated and diluted with a phosphate buffer. The samples were then delipidated, injected with isotopically labeled standards, and hydrolyzed for 24 h. Subsequently, the processed plasma samples underwent liquid chromatography-electrospray ionization tandem mass spectrometry. Additional oxidative stress markers 8-IP and 8-OHdG were measured in urine samples at Cayman Chemical (Ann Arbor, MI). 8-IP was quantified using affinity column chromatography followed by an enzyme immunoassay, and 8-OHdG was measured directly through an enzyme immunoassay⁴⁸ .

The cytokines IL-1β, IL-6, IL-8, IL-10, and TNFα were quantified using the Milliplex MAP High Sensitivity Human Cytokine magnetic bead panel (EMD Millipore Corp., St. Charles, MO, USA) with plates read on a Luminex 200 system using xPonent software, version 3.1 (Luminex Corporation, Austin, TX, USA). 50 μL of plasma was analysed per well, and all samples were assayed in duplicate as per the manufacturer's recommendation. Normalisation of data from different assay plates was performed by Merck Millipore (Frenchs Forest, NSW, Australia). Data for IL-10 was subsequently excluded from analyses due to excessive variance from the reported sensitivity and performance of the IL-10 assay. To confirm the accuracy of the Luminex system, IL-6 results were compared with those obtained using a quantitative enzyme immunoassay (Quantikine HS; R&D Systems Inc., Minneapolis, MN) and found to be highly correlated (P < 0.0001). von Willebrand factor (vWF) activity was measured with the ATA-Liatest vWF kit (Diagnostica Stago, Parsippany, NJ) and Diagnostica Stago STAR-automated coagulation analyser. High sensitivity C-reactive protein (hsCRP) levels were determined with an immunonephelometric method (Dade Behring Marburg GmbH, Marburg, Germany).