Tunel cell apoptosis detection kit

After the primary neurons were transfected and then treated with OGD/R, the cells were fixed with 4% paraformaldehyde at room temperature for 20 min, washed with PBS for three times each time for 5 min. According to the instructions of the TUNEL cell apoptosis detection kit (Jiangsu KeyGEN BioTECH Corp., Ltd., Nanjing, China), corresponding reagents were successively added and cells were photographed under a fluorescence microscope (Olympus). The experiment was repeated for three times.

Flow cytometry analysis for cell apoptosis was performed using an Annexin V-PE/7-AAD Apoptosis Detection Kit (Vazyme, A213-01). In H₂O₂-induced apoptosis experiments, cells were treated with H₂O₂ with a final concentration of 400 μM for 4 h before apoptosis detection. Cells were digested with ethylenediaminetetraacetic acid (EDTA)-free trypsin (Macgene, CC035) for 3 min, collected by centrifugation, washed with ice-cold phosphate-buffered saline (PBS), and resuspended at a density of 5 × 10⁵ cells/ml with 100 µl 1× binding buffer. Then 5 μl Annexin V-PE and 5 μl 7-AAD were added and incubated for 10 min in the dark. Finally, cells were incubated with an additional 400 µl 1× binding buffer and analyzed within 20 min by CytoFLEX S (Beckman Coulter Life Science). At least 1 × 10⁴ cells were collected to determine the percentage of apoptotic cells. CytExpert (version 2.4) was used to analyze flow cytometry data. For TUNEL assays, the TUNEL Cell Apoptosis Detection Kit (KEYGEN, KGA703) was used according to the manufacturer’s protocol.

Apoptotic hepatocytes were detected with TUNEL cell apoptosis detection kit (Biotin tagged POD method, KeyGEN BioTECH Corp., Ltd., Jiangsu, China) in paraffin-embedded section following the manufacturer's instructions.