Isolation and Knockdown of SLAMF7 in CD14+ Myeloid Cells

CD14+ myeloid cells (i.e. monocytes and macrophages) were isolated from human atherosclerotic plaques using magnetic beads bound to anti-CD14 antibody (Invitrogen, USA), as previously described [32 (link)]. Alternatively, peripheral blood monocytes (PBMCs) were separated by density gradient centrifugation of healthy donors. These cells were cultured overnight in fully supplemented DMEM in a 37°C 5% CO₂ incubator. siRNAs targeting SLAMF7 (Invitrogen, USA) and HiPerFect transfection reagent (Qiagen, USA) were used to knockdown the protein expression of SLAMF7 in the cultured CD14+ cells. Conditioned media was collected from cultured cells, stimulated with 10 ng/mL of LPS for 24h [33 (link)]. Human aortic smooth muscle cell line was purchased from LifeLine Cell Technology (Shanghai, China).