Annexin 5 fitc and pi apoptosis detection kit

The effect of miR-143 expression on NCI-H23 cell proliferation was assessed using CCK-8 kit (Dojindo, Kumamoto, Japan). Briefly, 5 × 10³ transfected cells were seeded in 96-well plates and cultured for 48 h. Exactly 10 μL WST-8 was added to each well and further incubated for 1 h at 37 °C. OD₄₅₀ was measured using a microplate reader (Molecular Deviced, Sunnyvale, CA, USA).
Cells were washed with PBS and fixed with 70% ethanol. The percentage of apoptotic cells was measured by flow cytometry using the Annexin V/FITC and PI Apoptosis Detection Kit (Becton Dickinson, Cockeysville, MD, USA) according to the manufacturer instructions.

An Annexin V-FITC and PI apoptosis detection kit (556547, BD Biosciences) was used according to the manufacturer's directions. Briefly, cells were resuspended at 1 × 10⁵ cells/mL in 100 μL of binding buffer. Annexin V-FITC (5 μL) and PI (5 μL and 20 μg/mL) were added to the cell suspension, followed by incubation at room temperature for 10  minutes in the dark and then mixing with 1 mL of binding buffer. A total of 1 × 10⁶ cells/sample was analyzed on a FACSCalibur.

After transfection with miR-21-5p mimics, inhibitors, and negative control, respectively, for 12 h, cell apoptosis was evaluated by quantitative detection of externalized phosphatidylserine using an Annexin V/FITC and PI apoptosis detection kit (BD Biosciences, San Jose, CA). Cells were incubated with hemin for 18 h, washed twice with cold PBS solution and suspended in 1x binding buffer. The cell suspension (100 μl) was then incubated with Annexin V-FITC (5 μl) and propidium iodide (PI; 5 μl) for 15 min at 25°C. This was followed by addition of binding buffer (400 μl) to the cell suspension, and the samples were then analyzed within 1 h using a flow cytometer (BD, FACSAria, CA, United States).

The cell cycle and apoptosis were analysed by flow cytometry. After collecting the cells and washing them with cold PBS, a total of 1 × 10⁶ cells were fixed with 70% ice‐cold ethanol for 24 h at 4°C. The cells were resuspended in cold PBS and incubated with 50 μg/ml propidium iodide and 0.1 mg/ml RNase A at 37°C for 15 minutes. The DNA content of the A549 or H1703 cells was determined by BD FACS Calibur cytometry and analysed by ModFit LT software (Verity Software House). A549 or H1703 cells were incubated in FBS‐free medium for 24h to induce apoptosis. For the flow cytometry analysis, the A549 or H1703 cells were trypsinized and collected for the detection of apoptosis by using an Annexin V‐FITC and PI Apoptosis Detection Kit (BD Bioscience).