4.3.4. Apoptosis Analysis

The apoptosis assay was carried out using the Annexin V-FITC Kit and the manufacturer’s protocol from BD Biosciences. The 5 × 10⁵ cells/mL treated and untreated were suspended in cold binding buffer and stained simultaneously with 5 μL FITC-Annexin V (green fluorescence) and 5 μL PI in a dark at room temperature for 15 min. The percentages of apoptotic cells were determined by double staining with Annexin V-FITC/ PI. In each tube was added 400 μL of Annexin V binding buffer and the 5000 cells/sample were collected using FACSCantoII flow cytometer (Becton Dickinson—BD) and the analysis was performed using DIVA 6.2 software in order to identify early apoptosis (Annexin+/PI−), late apoptosis (Annexin+/PI+) and necrosis (Annexin−/PI+) [48].