Cell cycle detection kit

A transwell polycarbonate membrane cell culture insert (3.0 μm pore sized, Corning) was employed for the transwell assay, which allows the free diffusion of CAP nanospheres between the transwell insert and the bottom culture well, but not cell translocations.^{[28 (link)]} To quantitatively evaluate the release behavior of CAP@M, the CAP@M were seeded in the transwell inserts, with the CAP content of 20 μm, and placed on the top of the bottom wells, then incubated with fresh cell culture medium. At predetermined time points (from 0 to 36 h), cell culture medium (200 μL) in the bottom wells were collected, and equal volumes of fresh cell culture medium were replenished. The contents of released CAP nanospheres were determined by ICP-MS. To investigate the ability of CAP@M to induce cell apoptosis, block cell cycle and induce caspase-3 activation, the CAP@M was seeded in the transwell inserts, with the CAP amount of 5 and 20 μm, and then A549 cells were cultured in the bottom wells. After 24 h incubation, the A549 cells in the bottom wells were analyzed by Annexin V-FITC/propidium iodide apoptosis detection kit (Invitrogen, China), cell-cycle detection kit (Invitrogen, China), and Caspase-3 Activity Assay kit (Invitrogen, China) respectively, according to the manufacturer’s instructions, then analyzed by flow cytometry and fluorescence microscopy.