We collected venous blood in heparinised tubes (Sarstedt, Germany) at day 3 after stroke onset. To avoid diurnal variation in cytokine production, we took blood between 7.00 and 7.30 am. Subsequently, we diluted the whole blood by 1:5 in sterile RPMI 1640 medium supplemented with l -glutamine (Sigma Aldrich, St. Louis, MO, USA) and we stimulated it in sterile tubes (Lonza, Walkersville, MD, USA) with lipopolysaccharide (LPS 10 ng/mL, E. coli 0111:B4, Sigma Aldrich, St. Louis, MO, USA) at 37 °C and 5% CO2. Based on previous publications [9 (link), 15 (link)], we stimulated blood for 4 h for TNFα and IP-10 and 24 h for interleukin IL-1β, IL-6, IL-8, IL-10, and IL-12p70. We removed supernatants and stored them at − 80 °C.
To measure concentrations of TNFα and IP-10, we used commercially available ELISA kits from R&D Systems (Minneapolis, MN, USA). We measured IL-1β, IL-6, IL-8, IL-10, and IL-12p70 concentration using a cytometric bead array immunoassay (Human Inflammatory Kit, BD Bioscience, San Diego, CA, USA). To measure the level of IL-6 in plasma, we used ELISA kits from R&D Systems (Minneapolis, MN, USA) with a detection limit of 0.11 pg/ml.
To measure concentrations of TNFα and IP-10, we used commercially available ELISA kits from R&D Systems (Minneapolis, MN, USA). We measured IL-1β, IL-6, IL-8, IL-10, and IL-12p70 concentration using a cytometric bead array immunoassay (Human Inflammatory Kit, BD Bioscience, San Diego, CA, USA). To measure the level of IL-6 in plasma, we used ELISA kits from R&D Systems (Minneapolis, MN, USA) with a detection limit of 0.11 pg/ml.
Free full text: Click here
