Cd14 mab coated micro beads

A sample of venous blood (40 mL) was obtained from 61 healthy controls selected according to the rs7044343 polymorphism (21 with CC, 21 with TC and 21 with TT genotypes). The peripheral blood mononuclear cell (PBMC) population was isolated by gradient centrifugation on Lymphoprep (Axis-Shield PoC AS, Oslo, Norway). Monocytes were isolated by positive selection with CD14-mAb-coated micro beads (Miltenyi Biotec, Bergisch Gladbach; Germany) following the manufacturer’s instructions (purity of 95–98%).

Whole blood collected in EDTA tubes was diluted 1:1 with 1X PBS–1% heparin and subsequently added to a Ficoll-Histopaque solution (Sigma, Sigma, St. Louis, MO, USA, Catalog Number 10771). The samples were centrifuged at 1300 rpm for 30 min, and the peripheral blood mononuclear cells (PBMCs) were generated after the centrifugation. Then, positive selection to obtain monocytes was carried out using CD14-mAb-coated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany; Catalog Number 130-050-201) following the manufacturer’s instructions (purity of 95 ± 98%). An aliquot of 1 mL of Tripure TM reagent (Roche Life Science, Penzberg, Germany) was added for collecting the monocytes. The cells were stored at −80 °C.