Annexin 5 fitc solution

Annexin V-FITC and propidium iodide (PI) double-staining with flow cytometry was performed for cell apoptosis detection. In brief, 3×10⁵ cells per well were plated onto 6-well plates, then grown overnight at 37 ˚C with 5% CO₂ for 24 h. Post 48 h transfection as above, the cells were collected and centrifuged for 5 min at 1,000 rpm, then resuspended in 195 μL 1×Binding Buffer (Sigma-Aldrich, USA), followed by 5 μL Annexin V-FITC solution (Sigma-Aldrich, USA) was added per sample and incubated at room temperature without lighting for 10 min. After being centrifuged at 1,000 rpm for 5 min, the cells were resuspended in 190 μL 1×Binding Buffer, followed by 10 μL PI (Sigma-Aldrich, USA) was added per sample. Subsequently, the stained cells were detected and analyze by a BD FACSCanto II flow cytometer (BD, USA).

After culturing for 5 d, hippocampal neurons were plated in six-well plates with paclitaxel at final concentrations of 0, 0.01, 0.1, 1 and 10 µmol/L. After 24 h of treatment, cells were digested by pancreatin and incubated with annexin V-FITC solution (Sigma, St. Louis, MO) for 15 min in the dark. Subsequently, cells were stained with propidium iodide (PI) and annexin V-FITC and analyzed on a FACScan flow cytometer (Sigma, St. Louis, MO). The excitation wavelength was 488 nm, and the fluorescence of FITC and PI was measured at 515 and 530 nm, respectively.

Analysis of the cell cycle is a key test for determining each phase cell population when treated with cytotoxic substances. Apoptosis rate in the colon cancer cells (HCT116) was quantified using annexin V-FITC (V-fluorescein isothiocyanate) (BD Pharmingen, San Diego, CA, USA). Cells were transferred to 6-well culturing plates (3–5 × 10⁵ cells per well) and incubated overnight. Cells were then treated with nitazoxanide for 48 h. Next, media supernatants and cells were rinsed with ice-cold phosphate-buffered saline (PBS). The next step was suspending the cells in 100 µL of annexin binding buffer containing 1.4 M NaCl, 25 mM CaCl₂ and 0.1 M HEPES/NaOH (final pH equals 7.4) and then incubated with 1:100 5-FU annexin V-FITC solution and 10 µg/mL propidium iodide (PI; Sigma, St. Louis, MO, USA) in the dark for half an hour. Stained cells were then measured by a Cytoflex flow cytometer (Beckman Coulter Inc., California, USA). Data were analyzed using cytExpert software (V2.4) [50 (link),51 (link)].