Annexin 5 fitc pi apoptosis detection kit

ARPE-19 cell line was purchased from the China Center of Type Culture Collection (Shanghai, China). DMEM medium was obtained from Viva-Cell (Shanghai, China). Fetal bovine serum (FBS) was purchased from Si-Ji-Qing (Hangzhou, China). Sodium hydrosulfide (NaHS, 68%∼72%) was obtained from Macklin (Shanghai, China). LED blue light (435–445 nm, 11.2 W/cm²) device was purchased from JDL company (Hangzhou, China). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-GRP78, anti-CHOP and anti-β-Actin antibodies were purchased from Beyotime (Shanghai, China). Annexin V-FITC/PI apoptosis detection kit was purchased from Beyotime (Shanghai, China). Superoxide dismutase (SOD) and malondialdehyde (MDA) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 3-(4,5-dimethylthiazol-3-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased form Beyotime (Shanghai, China). TUNEL kit, Antigen Repair kit and FAS eyeball fixative were purchased from Servicebio (Wuhan, China). Other common reagents used in the study are of analytical purity grade.

The Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime, Shanghai, China) was used to analyze the apoptosis of SW620 cells after SS treatment. Cells (2.5 × 10⁵) were inoculated in 6-well plates overnight, drugs were added, and culture was continued for 24 h. Cells were collected and washed twice with pre-chilled PBS. The cells were then treated according to the instructions of Annexin V-FITC/PI Apoptosis Detection Kit, and apoptosis was detected using flow cytometry (Beckman, Shanghai, China).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma Aldrich (USA). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA (0.05%), penicillin-streptomycin (P/S), PBS, calcein-AM, and enhanced chemiluminescence (ECL) western blotting detection reagent were purchased from Thermo Fisher Scientific (USA). GE11 polypeptide was purchased from GL Biochem Ltd. (Shanghai, China). Radioimmunoprecipitation assay (RIPA) lysis buffer, the primary antibodies of anti-PI3K (4292s), anti-p-PI3K (4228s), anti-Akt (9272s), anti-p-Akt (4060s), anti-Bcl-2 (3498s), and anti-Bax (5023s), were purchased from Cell Signaling Technology (USA). Bio-Rad protein assay kit and 30% acrylamide/bissolutionandpolyvinylidene difluoride (PVDF) membrane were obtained from Bio-Rad (USA). Annexin V-FITC/PI apoptosis detection kit, reactive oxygen species (ROS) detection kit, paraformaldehyde, 4,6-diamidino-2-phenylindole (DAPI), and Tubulin-Tracker were purchased from Beyotime Institute of Biotechnology (Shanghai, China).

Detection of apoptotic cells was performed using the Annexin V-FITC/PI apoptosis detection kit (Beyotime Biotech, Shanghai, China) according to manufacturer’s instructions. Briefly, cancer cells treated with 4, 6, and 8 μg/ml of morusin, respectively, for 36 h were washed with PBS and stained simultaneously with FITC-conjugated Annexin V and PI at room temperature for 15 min in the dark. The apoptotic cells were measured using a FACScalibur flow cytometer and Cell Quest Pro software (BD Biosciences, Shanghai, China). Three independent experiments were performed.