Annexin 5 fitc

For apoptosis assays, colon cancer cells were seeded at 1× 10⁵ per well in 12-well plates and cultured for 24 hours. On the second day, cells were transfected with siControl or siChREBP. After 48 hours, cells were harvested with trypsin and washed 2 times in ice-cold PBS, resuspended in 100 μl of binding buffer and incubated with Annexin V-FITC (4 A Biotech, China) for 5 min at 4 °C in the dark. After staining, the cells were incubated with propidium iodide for 5 min at 4 °C in the dark and then assayed with a flow cytometer (Beckman Coulter, Germany)^{42 (link)}. Software FlowJo (Becton, Dickinson & Company, USA) was used to analyze cell apoptosis.

Cells were transfected with siRNA for 48 h, after which they were collected and centrifuged. Subsequently, the medium was removed and the cells were stained with annexin V/FITC and 7AAD (4 A Biotech). Finally, the cells were stored in the dark at room temperature for 15 min. Flow cytometry analyses were conducted by utilizing a FACSCalibur flow cytometer (BD Biosciences) to identify cell apoptosis. FlowJo software was used to analyze the data.

Six-well plates (Corning, New York, United States) were seeded with 16-HBE cells at a concentration of 2 × 10⁵ cells per well. At 24 h after seeding, cells were treated with vehicle or test compounds and incubated for 24 h. Cells were then digested with trypsin to prepare single-cell suspensions. The digests were centrifuged and resuspended in antibody-binding buffer, then the cells were counted. Annexin V-FITC (4A biotech, Suzhou) (5 mL per sample) was added to each sample of 1 × 10⁵ resuspended cells and mixed gently. Samples were incubated for 10 min in the dark at room temperature. After centrifugation, the supernatant was discarded, and cells were resuspended in 100 mL of antibody binding buffer. Propidium iodide staining solution (4A biotech, Suzhou) (10 mL) was added to each sample and gently mixed well, then staining was immediately halted by adding PBS. Flow cytometry was performed immediately (Beckman Coulter, United States).