Caspase-3/7 activity assay and cell apoptosis analysis

The cells were seeded in a 96-well plate with 10⁴ cells/well, with a blank control well without cells. Then, Caspase-Glo (G8091, Promega) was added to each well, shaken for 30 min and incubated for 2 h at room temperature. The Caspase-Glo 3/7 assay releases a substrate of luciferase after being cleaved by caspase-3 and -7. The substrate of luciferase results in a luminescent signal that can be detected by a Synergy 2 Multi-Mode Reader (BioTek).

Annexin V-APC (88-8007-72, eBioscience) was used for cell staining in the cell apoptosis assay. According to the manufacturer’s instructions, the cells (≥5 × 10⁵/well) were washed, digested and then resuspended using 200 µl 1× binding buffer. Then, 10 µl annexin V-APC was added to 200 µl cell suspension and incubated in the dark at room temperature for 10–15 min. Cell apoptosis analysis was detected by a Guava easyCyte HT cytometer (Millipore).