Ab181416

A total of 10 mL of venous blood was obtained from a peripheral vein and was subjected for centrifugation and subsequent plasma separation and eventual central storage at −80°C for further analysis of MCP-1, TNF-alpha, IL-6, leptin, adiponectin, and IL-10. Another 10 mL of the venous blood sample was collected into EDTA tube and stored at room temperature. It was used for determining the total and differential leukocyte count (TLC and DLC), CD4+ T-lymphocytes, and its populations and cell immune markers (CD4⁺ T cell and its subpopulations) using flow cytometry. Plasma samples were used for biochemical analysis, including glucose, lipid profile, liver, kidney functions, hormonal parameters (c-peptide and insulin), and were performed at HMC labs. Assessment of MCP-1, TNF-alpha, IL-6, leptin, adiponectin, and IL-10 was performed in the Biomedical Research Center (BRC) labs at Qatar University using Luminex 200 by the multiplex technique according to the manufacturer’s protocol. Adipokines and cytokines were measured by the Multiplex Elisa technique using magnetic beads (HADK2MAG-61K, MADCYMAG-72K from Millipore), IL-10 (MPXHCYTO-60K-06 from Millipore), and Elisa was used for hsCRP (ab181416 from Abcam). The assay was performed based upon the manufacturer’s protocol, as previously published by our lab.15 (link)

The blood samples of all patients and healthy controls were collected within 24 h after admission. The serum levels of HMGB1, I-FABP, and inflammatory factors CRP, IL-1β, IL-6 and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA) using commercial ELISA kits (HMGB1 LifeSpan Biosciences LS-F26519, I-FABP Abcam ab193700, CRP Abcam ab181416, IL-1β Abcam ab46052, IL-6 Abcam ab178013, and TNF-α Abcam, ab181421), according to manufacturer’s instructions.