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.