Annexin 5 fitc 7 aad

The cell apoptosis was measured using an Annexin V-FITC/7-AAD (#559763, BD Biosciences, NJ, USA) staining kit. HEI-OC1 cells were transfected with a REST plasmid or control vector, and then treated with H₂O₂. After treatment, cells were resuspended in binding buffer and stained with FITC-labeled Annexin V and 7-AAD, for 20 min at room temperature, in the dark. Cell apoptosis was detected using FACS Calibur (BD Biosciences, NJ, USA) and data were analyzed by using the FlowJo software v7.6.1 (FlowJo, LLC, NJ, USA).
Terminal Deoxynucleotidyl Transferase (TdT)-Mediated dUTP Nick-End Labeling (TUNEL) assay was used to detect the apoptotic cells, according to the manufacturer’s instructions. Cochlear sections were permeabilized in 0.3% Triton X-100 for 60 min, and then blocked with a fluorometric terminal deoxytransferase solution for 1 h, at 37 °C. The sections were then incubated with anti-Tuj1 primary antibody overnight at 4 °C. They were then stained with a Cy3-conjugated secondary antibody for 2 h, at room temperature. Finally, the nuclei were stained with DAPI, and fluorescent images were taken using a Leica TCS SP5 confocal fluorescent microscope.

For apoptosis analysis, KYSE-30 and TE-1 cells were seeded in 6-well plates at a concentration of 1 × 10⁶ cells/well with different agents for 48 h and divided into the four groups: control, anlotinib (20 μM), raltitrexed (2.5 μM) and anlotinib combined with raltitrexed (20 μM + 2.5 μM), the negative control for apoptosis analysis was the equivalent volume of DMSO. Apoptotic cells were distinguished by Annexin V-FITC/7AAD dual staining using apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Cells were collected and washed twice with cold PBS and centrifuged for 10 min at 20,000 rpm at 4 °C. Cells were cultured in 300 μL / 1 × 10⁶ cells of 1 × annexin-binding buffer at room temperature for 5 min to prepare the cell samples for flow cytometry, and then double stained with annexin V-FITC (green) and 7AAD-PerCP (red) for 15 min. The percentage of apoptotic cells was analyzed with a FACS Calibur flow cytometer (BD Biosciences) using Flow Jo software (version 9.8.1). The apoptosis rate in every group was described by Q2 (late apoptosis) + Q3 (early apoptosis). All experiments were repeated for three times.