Figure 2.

Triple-cell co-culture provides pure cellular niches for macrophages, vascular endothelial and vascular smooth muscle cells. (A) Prior to completion of co-cultures via the addition of THP-1 cells, EC/SMC coated transwell inserts were analyzed via immunofluorescent microscopy for CD31 (red) and αSMA (green). Scale Bars = 40 μm. Following completion of EC/SMC/THP-1 co-cultures using both 0.4 μm (B) and 3 μm (C) transwell inserts, cells were isolated from each cellular niche and analyzed by immunoblotting for eNOS, αSMA, and CD11b. n = 3 co-cultures. (D) SMC were cultured on the underside of 3 μm transwell inserts and 3 days later analyzed by immunofluorescent microscopy for the expression of αSMA (green). (E) ECs or SMCs were cultured on 0.4 μm inserts until confluent. They were then mechanically disrupted and analyzed for viability using trypan blue exclusion. n = 6 cultures. Scale Bar = 10 μm.