ML221

The proliferation of bEnd.3 cells following 24 h incubation with ML221 (0-30 μM) are assessed by the MTT assay and the BrdU incorporation assay. Mouse endothelial cell line bEnd.3 cells are maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum. The cells are plated at a density of 2.5 × 10⁴/well in 24 well culture plates for the MTT assay and the BrdU incorporation assay. To perform the BrdU incorporation assay, 10 μM BrdU is added to the culture medium. After 2 h, the cells are fixed with 4% paraformaldehyde for 10 minutes. After removing the 4% paraformaldehyde, the cells are incubated with 2 M hydrochloric acid for 10 min at 37℃ for DNA hydrolysis, followed by neutralization with 0.1 M Sodium Borate buffer (pH 8.5) for 30 min at room temperature. Intranuclear BrdU is labeled with mouse anti-BrdU antibodies. The primary antibody is visualized with biotinylated goat anti-mouse IgG antibodies plus streptavidin fluorescein isothiocyanate. Nuclei are detected with Hoechst33342. The BrdU incorporation rate is determined by calculating the numbers of BrdU positive cells per Hoechst positive cells.