Annexin 5 fitc propidium iodide detection kit

Instruments, reagents, and the experimental animals were provided by the animal center of Tongji Medical College and Huazhong University of Science and Technology. IL-1β was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Icariin (purity ≥ 98%) was purchased from Nanjing Zelang Pharmaceutical Technology (Nanjing, China). Fetal bovine serum was purchased from Gibco. F12-Dulbecco's modified Eagle medium was purchased from Hyclone (Logan, UT, USA). Cell counting kit-8 (CCK8) was purchased from Kaiji Bioengineering Institute (Jiangsu, China). LY294002 was purchased from Sigma-Aldrich (St. Louis, MO, USA). The reactive oxygen species (ROS) detection kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). JC-1 assay kit was purchased from Beyotime (Beijing, China). Annexin V-FITC/propidium iodide detection kit was purchased from Nanjing KeyGen Biotech (Nanjing, China). β-Actin, Bcl-2, bax, caspase-3, phospho(p)-AKT, rabbit monoclonal antibodies, and the p53 and AKT mouse monoclonal antibody were purchased from Abcam (Cambridge, UK). Goat antirabbit and goat antimouse IgG were purchased from Proteintech (Wuhan, China). Microplate reader was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The inverted fluorescence microscope used was manufactured by Olympus (Japan).

Instruments, reagents, and experimental animals were provided by the animal center of Tongji Medical College and Huazhong University of Science and Technology. H₂O₂ was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Icariin (purity ≥ 98%) was purchased from Nanjing Zelang Pharmaceutical Technology (Nanjing, China). Fetal bovine serum was purchased from Thermo Fisher. F12-Dulbecco's modified Eagle medium was purchased from HyClone (Logan, UT, USA). Cell counting kit-8 (CCK8) was purchased from Kaiji Bioengineering Institute (Jiangsu, China). LY294002 was purchased from Sigma-Aldrich (St. Louis, MO, USA). The reactive oxygen species (ROS) detection kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Annexin V-FITC/propidium iodide detection kit was purchased from Nanjing KeyGen Biotech (Nanjing, China). β-Actin, Bcl-2, bax, caspase-3, phospho(p)-Akt, rabbit monoclonal antibodies, and the p53, Akt mouse monoclonal antibody, were purchased from Abcam (Cambridge, UK). Goat anti-rabbit and goat anti-mouse IgG were purchased from Proteintech (Wuhan, China). Microplate Reader was purchased from Thermo. Inverted fluorescence microscope was from Olympus, Japan.

Apoptosis analysis was performed using the annexin V–FITC/propidium iodide (PI) Detection Kit (KeyGen Biotech, KGA108) (following the manufacturer’s protocol). Cells were harvested and washed three times with cold PBS. After cells were resuspended in 500 μl binding buffer, 5 μl PI and 5 μl annexin V-FITC were both added into the cell suspension. At least 10,000 live cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences). FlowJo software was used to analyze the data.
Intracellular ROS content was detected using the fluorescent probe DCFH-DA. Inoculate an appropriate amount of cells in a six-well plate, and treat them with GYZ for 48 h after adherence. Replace the old medium with double-free medium, add 5 μM DCFH-DA to each well, and place it in the cell culture incubator for 30 min. Cells were washed with cold PBS for three times. After cells were resuspended, FACSCalibur flow cytometer (BD Biosciences) was used to measure the fluorescence intensity. FlowJo software was used to analyze the data.