Multiplex Protein Biomarker Profiling in Plasma

Whole blood was collected in heparin tubes, centrifuged at 2500 rpm for 10 min for plasma separation and stored at −80 °C. Plasma samples tested in different batches were diluted 1:2, and the levels of plasma biomarkers were measured using commercial kits, Human Magnetic Luminex assays (Lot number L129636 R&D, Minneapolis, MN, USA), as previously published [29 (link)]. The kit detected 15 soluble proteins, namely, IFN-gamma, NCAM, TNF-alpha, IL-8, IL-10, IL1b, GM-CSF, IL-13, IL-12, CD40-ligand, MMP-1, MMP-2, MMP-8, S100A8 and MPO. The sample plates were read on the same day using a MAGPIX system, xMAP instrument (Luminex, Austin, TX, USA). Finally, xPONENT software, version 4.3 (Luminex, Austin, TX, USA), was used for bead acquisition and analysis.