Apoptosis Assessment in hBMECs

Morphological assessment of apoptotic cells was processed by Hoechst 33258 staining method as described earlier (Yang et al., 2013 (link)). Briefly, hBMECs were seeded in 6-well culture plates at a density of 1 × 10⁵ cells/well and treated with various concentrations of AS (25, 50, and 100 μM), TAK-242 (1 μM), or same volume of serum-free DMEM (vehicle control) for 12 h, then treated with Aβ_1-42 (50 μM) or medium for another 24 h at 37°C. After incubating with fixative solution for 10 min, the medium was removed and washed with PBS for 15 min. Then, the cells were treated with 500 μL staining solution of Hoechst 33258 (10 μg/ml) for 5 min, followed by washing with PBS for 15 min at a dark room for reducing the background. Finally, the nuclear morphological changes of apoptotic cells were observed under a fluorescent microscope at ×400 magnifications (AMG EVOS, Thermo Fisher Scientific, Inc., Waltham, MA, United States) and the percentage of apoptotic cells was calculated according to the ratio of apoptotic cells to total cells. All experiments were performed three times in triplicate.