Cell apoptosis was evaluated using Annexin V-FITC and Propidium Iodide (PI) Kit (Sigma, USA) by flow cytometry as previously described [13 (link)]. After washed with PBS and resuspended with binding buffer, 5 μl Annexin V-FITC and 5 μl PI were added to the 1000 μl cell resuspension with 1 million cells (in Annexin-V binding buffer) and incubated for 30 mins in the dark. Stained cells were centrifuged and washed twice with Annexin-V binding buffer and resuspended in 400 μl Annexin-V binding buffer. The percentage of apoptotic cells was detected by a flow cytometer (Becton Dickinson).
To determine the cellular ROS level, cells with different treatments were stained with 2 µM CM-H2DCFDA (Thermo Fisher Scientific, CA, USA) at room temperature for 30 mins. Cells were then washed once and resuspended in PBS for flow cytometry analysis in FITC channel.
To determine the cellular ROS level, cells with different treatments were stained with 2 µM CM-H2DCFDA (Thermo Fisher Scientific, CA, USA) at room temperature for 30 mins. Cells were then washed once and resuspended in PBS for flow cytometry analysis in FITC channel.