2.7. The Analysis of Cell Apoptosis via Annexin and Flow Cytometry

The apoptosis level was analyzed with an annexin V-FITC/PI apoptosis kit as per the instructions of the manufacturer. The ARPE-19 cells were inoculated into the culture plate at 1 × 10⁵ cells/ml and subjected to treatment with and without CMCT, as well as H₂O₂ for 12 h. The collected cells were rinsed two times using cold PBS, after which they were resuspended in a new medium and stained at room temperature for 15 min away from light with annexin V-FITC/PI. Then, they were assessed via flow cytometry (BD Bioscience, USA), while a Cell Quest analysis tool (BD Biosciences, Franklin Lakes, NJ, USA) was used to calculate the degree of apoptosis. The results were as follows: normal cells: annexin-V-negative-PI-negative; early apoptotic cells: annexin-V-positive-PI-negative; late apoptotic or necrotic cells: annexin-V-positive-PI-positive. All experiments were repeated three times.