Annexin 5 fluorescein isothiocyanate fitc propidium iodide apoptosis kit

Na¹³¹I was purchased from Shanghai XinKe Pharmaceutical Co., Ltd. ²²³RaCl₂ was purchased from the Institute for Energy Technology. The operation related to the above agents was performed in a hot cell. Ethiodized poppyseed oil injection was purchased from Jiangsu HengRui Medicine Co., Ltd. Silk fibroin microspheres (about 150.61 ± 10.39 μm in diameter, controlled by steel screens) were purchased from Suzhou Meilun Biotechnology Co., Ltd. The TdT-mediated dUTP Nick-End Labeling (TUNEL) kit was purchased from KeyGEN Biotechnology Co., Ltd. The annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit was purchased from BD Bioscience. A cell counting kit-8 (CCK-8) was purchased from the Beyotime Institute of Biotechnology. A γ-H2AX ELISA kit was purchased from Jiangsu Meimian Industrial Co., Ltd.
A human hepatocyte carcinoma cell line (Huh-7) was purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The New Zealand white rabbits (male, weighing 2.5 ± 0.5 kg) were purchased from Ailingfei Biotechnology Co., Ltd., and kept under a specific pathogen-free condition at the laboratory animal center. This research was approved and guided by the Ethics Committee of Naval Medical University (Approval No. 1207050662).

The fluorescence-activated cell sorting (FACS) technique was employed to analyze the 3c- and 3e-induced cell death, the activation of executive caspase-3, and the cell cycle in the A549 cells. The quantitative analysis of apoptosis and necrosis was performed using an Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) apoptosis kit (BD Biosciences, BD Pharmingen™, San Jose, CA, USA) as previously described [36 (link)]. To determine the active form of caspase-3, the A549 cells were plated into 6-well plates at a density of 6 × 10⁵ cells/well. The next day, the growth medium was replaced with a fresh one supplemented with compound 3c or 3e (10, 20, and 40 µM) or the DMSO vehicle (0.1%; control cells). After 48-h incubation, the samples were harvested and the level of active caspase-3 was determined using the phycoerythrin (PE) Active Caspase-3 Apoptosis Kit (BD Pharmingen™) according to the manufacturer’s instructions. The stained cells were analyzed using FACS Calibur, and data were analyzed using Cell Quest Pro Version 6.0 (BD Biosciences, San Jose, CA, USA) for the Macintosh operating system. The results were calculated as a percent of cells with active caspase-3 among all the analyzed cells. The cell cycle analysis was performed by determination of the DNA contents on the basis of PI staining as previously described [36 (link)].

Each cell was treated with the indicated doses of QS1189 for 24 h. To validate the cell cycle, cells were fixed in 70% ethanol at −20 °C from 1 h to a few days, incubated with 5 μL RNase (10 mg/mL), and finally stained with 10 μL propidium iodide (1 mg/mL). Cellular DNA content in the treated cells was analysed with a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Apoptosis was quantified using the annexin V-fluorescein isothiocyanate (FITC)/propidium iodide apoptosis kit (BD Biosciences) in accordance with the manufacturer’s protocols. Cells were resuspended in annexin V-binding buffer (150 mM NaCl, 18 mM CaCl₂, 10 nM HEPES, 5 mM KCl, and 1 mM MgCl₂). FITC-conjugated annexin V (1 μg/mL) and propidium iodide (50 μg/mL) were then added to the cells and incubated for 30 min at room temperature in the dark. Analyses were performed using an FACScan flow cytometer. Data were analysed with CellQuest software (BD Biosciences).