2.11 | Macrophage adhesion assay
Bone marrow-derived mononuclear cells were isolated from the femurs of WT mice and cultured in high-glucose DMEM supplemented with 10% FBS and 20% L929-conditioned medium for 5 days; at this point, adherent mononuclear cells are macrophages. SKI SMCs were pretreated with PDTC (10 μM) or pioglitazone (10 μM) for 48 hr before macrophage adhesion assay. DMSO acted as solvent control. For macrophage adhesion assay (Tong et al., 2016), SMCs were plated in 12-well plate, at a density of 106 cells/well, in 0.2% FBS DMEM until confluent. Macrophages (5 × 105 cells/well) were added to SMCs and incubated for 1 hr; the media was removed and SMCs washed 3 times with PBS to remove unbound macrophages. Four images were taken in each well and the number of bound macrophages was counted per image area.