7 aad

For analysis of apoptosis, IEC-6 cells were fixed and stained with PE Annexin V Apoptosis Detection kit with 7-AAD (Vazyme, A213-01) according to the manufacturer’s instructions. The proportion of PI-positive and PE-positive cells were recorded. Data acquisition was performed on Accuri C6 Plus and analyzed via FlowJo (RRID:SCR_008520).

The apoptosis of apoptotic cells was assessed by flow cytometry. 2 × 10⁵ cells were seeded overnight in 6-well plates and then treated with IR for 24 h. Thereafter, the cells were collected, washed three times with cold PBS, stained with Annexin V-PE and 7-AAD (Vazyme, Nanjing, China) in binding buffer for 10 min at room temperature and in the dark and then quantified with a BD FACSCalibur flow cytometer (BD Sciences, USA).

The cell cycle distribution and apoptotic cell population were scrutinized as described previously (23 (link)). In brief, for the cell cycle, collected cells were fixed with 70% ethanol (Sangon Biotech, Shanghai, China) and stained with 0.5 ml propidium iodide (PI) (Beyotime Biotechnology, Shanghai, China) for 30 min. For cell apoptosis analysis, collected cells were stained with 5 μL Annexin V-PE and 5 μL 7-AAD for 10 min (Vazyme, Nanjing, China). The cells were examined using flow cytometry (BD, New Jersey, USA). The cell cycle data were analyzed with FCS Express Launcher software (De Novo Software), and the cell apoptosis results were analyzed with FlowJo software.