Pi annexin 5 solution

For flow cytometry, 2 mL/well cells (about 2 × 10⁵) were incubated in 6-well plates and treated according to the protocol. The cells were digested and washed twice with phosphate-buffered saline (PBS) and then fixed with 75% ethanol overnight at 4 °C. Then, the cells were washed three times with PBS, and incubated with propidium staining and RNase A (Beyotime, Shanghai, China) at 37 °C for 30 min. Then, the cell cycle was analyzed using a flow cytometer (Beckman Coulter, Brea, CA, USA). The cells were harvested and washed once with PBS and incubated for 20 min at 37 °C with 10 μM of 2,7-dichlorofluorescein diacetate (Beyotime, Shanghai, China). After being washed with PBS three times, the production of reactive oxygen species (ROS) was analyzed using a flow cytometer. The cells were collected and stained with 500 µL of 1 × JC-1 dye solution (Beyotime, Shanghai, China) at 37 °C for 20 min in the dark. Then, the cells were washed twice and resuspended using a 1 × JC-1 staining buffer, and the mitochondrial membrane potential (∆Ψm) was analyzed using flow cytometry. The cells were digested and washed once with PBS and then resuspended in a PI/Annexin-V solution (KeyGEN, Jiangsu, China). Finally, the apoptosis rate was analyzed using a flow cytometer.

Cells were cultured and exposed to the tested compounds for 24 h, harvested by trypsinization, and washed with PBS, then resuspended and incubated in PI/Annexin-V solution (KeyGEN Biotech) for apoptosis analysis. At least 10,000 live cells were analyzed on a FACSCalibur flow cytometer. Data were analyzed using FlowJo7.6.1 software.