Annexin 5 fitc pi cell apoptosis detection kit

Mesoporous silica nanoparticles were purchased from Anhui JingYe Nano Technology Co., Ltd. N-(trimethoxysilylpropyl) ethylenediamine triacetic acid was obtained from J&K Scientific Ltd. Doxorubicin hydrochloride was obtained from Shenzhen Wanle Pharmaceutical Co., Ltd. N-hydroxysuccinimide (NHS) was purchased from Aladdin Industrial Corporation. Amine-Peg2000-Biotin, the EZ-Link™ Sulfo-LC Biotinylation Kit and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Thermo Fisher Scientific. Dylight 488-Avidin was obtained from Wuhan Boster Biological Engineering Co., Ltd. Rituximab was obtained from Hoffmann-La Roche, Ltd. The DAPI (4,6-diamidino- 2-phenylindole) staining solution, Cell Counting Kit-8 and the Annexin V-FITC/PI cell apoptosis detection kit were purchased from Beyotime Biotechnology Co., Ltd.

The Annexin V–FITC/PI cell apoptosis detection kit (Beyotime, Shanghai, China) was used to determine the apoptosis rate of the A549 cells. The experimental operation was carried out according to the operating instructions provided by the reagent kit manufacturer. The flow cytometry was used to detect the cell apoptosis rates.

The Annexin V/propidium iodide (PI) double-staining assay was employed to detect cell apoptosis. HepD cells (2 × 10⁵ cells/well) were seeded in the laser confocal glass-bottom culture dish and incubated for 24 h. Then, cells were treated with the Im and La mixture (0, 20, 50, 75, and 100 μg/mL, respectively) for 24 h. According to the protocol of the Annexin V-FITC/PI cell apoptosis detection kit (Beyotime, Shanghai, China), Annexin V-FITC (10 μL) solution and PI (5 μL) dye were added to the cell culture dish and incubated for 30 min in the dark at 37 °C. Then, the fluorescence images were obtained with a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

The DNA contents of the cells were determined by flow cytometry to estimate the percentages of cells in different phases of the cell cycle. The treated cells were collected, washed twice with cold PBS, and fixed in 70% ethanol at −20 °C overnight. The cells were resuspended in PBS containing 50 mg/ml propidium iodide (PI) and 10 mg/ml DNase-free RNase immediately before flow cytometry. A FACSCalibur system (Becton Dickinson, San Diego, CA, USA) equipped with CellQuest software was used for flow cytometry analysis.
For apoptosis analysis, PASMCs were seeded at 3 × 10³ cells/well in 96-well plates using triplicate wells for each transfection. After 48 h, the cells treated under various conditions were incubated in 1.5% H₂O₂ for 24 h. The cells were then starved in medium containing 0.1% FBS; stained with Annexin V-FITC and PI reagents in binding buffer using the Annexin V-FITC/PI cell apoptosis detection kit (Beyotime Institute of Biotechnology, Beijing, China); and analyzed using a FACSCalibur flow cytometer.

DBCO-PEG₂₀₀₀-FA was purchased from Pengsheng Biotechnology Co., Ltd. DOTA-NHS was purchased from Xi’an Ruixi Biotechnology Co., Ltd. Silica gel and silica gel plates were purchased from Qingdao Ocean Chemical Engineering Co., Ltd. Lidocaine, propidium iodide, and sodium hydroxide were purchased from Shanghai Sigma Co., Ltd. Gadolinium (III) chloride hexahydrate (GdCl₃ 6H₂O) was purchased from Beijing Bailingwei Technology Co., Ltd. CCK-8 kit was purchased from Shanghai Macklin Co., Ltd. Folic acid (FA) and doxorubicin (DOX) were purchased from Shanghai Aladdin Co., Ltd. Calcein/PI Cell Viability Detection Kit, Annexin V-FITC/PI Cell Apoptosis Detection Kit, IL-6 ELISA Detection Kit, TNF-α ELISA Detection Kit, and Colony Formation Staining Solution were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Isoflurane was purchased from Shenzhen Ruiwode Life Technology Co., Ltd. Medical surgical glue was purchased from Beijing Shunkang Technology Development Co., Ltd. Cell culture medium, trypsin, ampicillin, and streptomycin sulfate were purchased from Hyclone Company. Human liver cancer cell lines (BEL-7402, Hep-G2, and HuH-7) and normal cell lines (HL-7702, HT-22, L929, IMR90, and HEK293) were all derived from American type culture collection (ATCC).

Apoptosis was detected using an Annexin V‐FITC/PI cell apoptosis detection kit (Beyotime), according to the manufacturer's instructions. Briefly, approximately 1 × 10⁶ cells were incubated with 5 μL Annexin V for 15 min at room temperature, followed by 10 μL PI (10 mg/mL) for 5 min at room temperature in darkness. Finally, the samples were analyzed using a flow cytometer (BD Biosciences).