MCP-1-primed macrophages show higher cytotoxicity.

(A) RAW macrophages (after 24 hours starvation) and SMCs were exposed to various culture conditions with or without addition of MCP-1 (100 ng/ml) for 24 hours. SMCs were also co-cultured with MCP-1-primed RAWs (100 ng/ml, 24 h), or naïve RAWs (PBS treated), or cell-free conditioned medium harvested from MCP-1-primed RAWs for 24 hours. Early apoptotic cells were identified as PE Annexin-V⁺/7-AAD⁻ by flow cytometric analysis. H₂O₂ (700 μM, 4 h) treatment was used as a positive control. (B) Co-culture was stained with the monocyte/macrophage marker CD11b prior to PE Annexin-V/7-AAD staining. (C) Representative bright field images of SMCs, RAWs, SMCs/RAWs co-culture (7 days), and SMCs/MCP-1 primed RAWs co-culture (7 days). Scale bar, 50 μm. Magnification, 20X. Data are mean±SEM. n = 3∼6, *p<0.05, **p<0.01 vs. untreated SMCs, #p<0.05, N.S.  =  not significant, One-way ANOVA.