Annexin 5 pi double staining

NP cell apoptosis was evaluated by flow cytometry using Annexin V/PI double staining according to the manufacturer’s instructions (Beyotime). Briefly, after NP cells were harvested as described above and washed with PBS, they were resuspended in 600 μL of Annexin V-FITC binding buffer. Then, 15 μL of Annexin V and 30 μL of PI solution were added to the NP cell suspension and incubated for 20 minutes at room temperature. Finally, the cell apoptosis rate was detected by flow cytometry (FACS Aria; BD Company) via the FACSDiva software (Becton Dickinson, Franklin Lakes, NJ, USA). These Annexin V positive-stained and PI-negative-stained cells and double positive-stained cells were regarded as dying cells in this assay.

After co-incubating with the conditioned medium for 48 h, apoptosis of HKFs was detected by annexin V/PI double staining (Beyotime, Shanghai, China). The operation was conducted based on the instructions. The BD Accuri C6 Plus Flow Cytometer (BD Biosciences, San Jose, CA, USA) was utilized for further analysis.