Annexin 5 fitc apoptosis detection kit

All of the chemicals were analytical grade reagents and without further purification. GdNO₃·6H₂O and catalase (CAT) from bovine live (≥40,000 units/mg protein) were purchased from J&K Scientific Ltd. (Beijing, China). Dopamine hydrochloride (DA), DCFH-DA, DAPI, Hoechst33342, and PI were supplied by Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Na₂WO₄·2H₂O was obtained from the Guoyao Chemical Research Institute (Shenyang, China). The HT29 cell line was obtained from the Cell Culture Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. 3-[4,5-di-methylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Shanghai, China). Annexin V-FITC apoptosis detection kit and Goat anti rabbit IgG-FITC were supplied by Absin Biotechnology Co., Ltd. (Shanghai, China). HIF-1α antibody and γ-H₂AX antibody were purchased from Cell Signaling Technology, Inc. (Danfoss, CA, USA). McCoy’s 5A medium was obtained from Biological Industries (Shanghai, China). Fetal bovine serum was purchased from Sigma-Aldrich (Shanghai, China).

According to the manufacturer’s instructions, the Annexin V-FITC Apoptosis Detection Kit (Absin, Shanghai, China) was utilized to evaluate the level of cell apoptosis. Initially, the cells were harvested and washed with cold PBS. Then, they were resuspended in 300 μL of binding buffer at a concentration of 10⁶ cells/ml. The cell suspension was then subjected to a 10-min incubation on ice in the dark with Annexin V-FITC, followed by a 5-min incubation with PI. Finally, the samples were analyzed using a DxP Athena™ Cytometer (Cytek Biosciences, USA), as previously described [42 (link)].

To identify the apoptotic rate, the percentage of cells undergoing apoptosis was determined by an annexin V-FITC apoptosis detection kit (Absin, Shanghai, China). HD11 cells were cultured in 6-well plates and infected with IBV at 10 MOI. Cells were harvested and washed three times with phosphate-buffered saline (PBS) at the indicated times. The cells were centrifuged at 500 g for 5 min and then suspended in 500 μL of binding buffer containing 5 μL FITC-conjugated annexin V antibody and 5 μL propidium iodide (PI). The mixture was incubated at room temperature for 15 min in the dark. The cells were detected by flow cytometer (BD Biosciences, San Jose, CA, USA) within an hour.