Annexin v 7 aad kit

The cell apoptosis rate was analyzed using an Annexin V/7-AAD kit (Biolegend, California, USA). Briefly, cells were treated with Res and collected by centrifugation. Then, they were resuspended in 500 μl binding buffer and mixed with 5 μl APC Annexin V as well as 5 μl 7-AAD staining solution. Finally, the mixture was incubated away from light at room temperature for 5 min, and cells were analyzed using flow cytometry (CytoFlex Beckman CytoFlex, USA).

Annexin V/7-AAD Staining: Cells were stained by using Annexin V/7-AAD kit (Biolegend, California, USA). Briefly, 200 µL cell suspension was centrifuged and the supernatant was removed. The pellet was resuspended in 100 µL Annexin V binding buffer (Biolegend, San Diego, CA, USA) and 5 µL Annexin V-APC (BioLegend, San Diego, CA, USA) and 7-AAD (Biolegend, San Diego, CA, USA) were added, followed by incubation for 15 min at room temperature in the dark. Measurement was performed on the LSRII (Becton Dickinson, Franklin Lakes, NJ, USA) flow cytometer using CellQuest analysis software (Becton Dickinson, Franklin Lakes, NJ, USA).
To analyze caspase-3 cleavage, cells were washed and then resuspended in 200 μL 4% paraformaldehyde for 15 min at room temperature in the dark. Cells were permeabilized in methanol and incubated on ice for 30 min. For immunostaining, cells were incubated for 1 h with Cleaved Caspase-3 rabbit mAb -AF647 (Cell Signaling, Cambridge, UK). Measurement was performed on the LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) using CellQuest analysis software (Becton Dickinson, Franklin Lakes, NJ, USA).