Sterile tubes

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.

The whole blood stimulation procedure was previously described^{30 (link)}. Venous blood was collected in heparinized tubes (Sarstedt, Nuembrecht, Germany) on Day 3 after stroke. To avoid diurnal variation, blood was obtained between 7:00 AM and 7:30 AM. The whole blood was diluted by 1:5 in sterile RPMI 1640 medium supplemented with L-glutamine (Sigma–Aldrich, St. Louis, MO). Then the samples were stimulated in sterile tubes (Lonza, Walkersville, MD) at 37 °C in 5% CO₂ with LPS (10 ng/mL, E. coli 0111:B4, Sigma–Aldrich, St. Louis, MO). Blood stimulation was performed for 4 h for TNFα and IP-10, and 24 h for IL-1β, IL-6, IL-8, IL-10, and IL-12p70. The supernatants were removed and stored at −80 °C until further analysis. TNFα and IP-10 concentrations were measured using a commercially available enzyme-linked imunoassay (ELISA) kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The levels of IL-1β, IL-6, IL-8, IL-10, and IL-12p70 were determined by a cytometric bead array (Human Inflammatory Kit, BD Biosciences, San Diego, CA).
Plasma levels of IL-6, soluble IL-6 receptor (sIL-6R), TNFα, and IL-1 receptor antagonist (IL-1ra) were measured using a commercially available ELISA kit (R&D Systems, Minneapolis, MN). The detection limit was 0.11 pg/mL for IL-6, 15.1 pg/mL for sIL-6R, 0.19 pg/mL for TNFα, and 18.3 pg/mL for IL-1ra.