Quantification of Apoptosis in Colon Cancer Cells

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)].