Cd14 micro magnetic beads

Peripheral blood mononuclear cells (PBMCs) were prepared on a Ficoll‐Hypaque density gradient centrifugation. CD14⁺ cells were obtained through positive selection by CD14⁺ micromagnetic beads according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, monocytes were differentiated in complete RPMI‐1640 medium supplemented with recombinant human macrophage colony‐stimulating factor (50 ng/mL; R&D Systems, Minneapolis, MN) for an additional 6 days before following study. Human primary aortic smooth muscle cells (HASMCs; ATCC PCS‐100‐012; American Type Culture Collection [ATCC], Manassas, VA) and human primary aortic endothelial cells (HAECs; ATCC PCS‐100‐011; ATCC) were cultured according to the manufacturer's instructions. Cells were used for experiments at passages 3 to 8.

Peripheral blood mononuclear cells (PBMC) from fertile women were isolated by density gradient centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden). CD14+ cells were separated by positive selection with CD14+ micro magnetic beads according to the manufacturer’s instructions (Miltenyi Biotec., Bergisch Gladbach, Germany). Cell population purity was checked by FACS analysis using anti-CD14 mAb and was found to be >95% for each set of experiments.