2.6. Apoptosis Detection

Cells were plated in 24 well-plates at a density of 3 × 10⁵ cells/mL and incubated for 24 h. After treatment with γ-tocotrienol, cells were collected and stained with 5 µL PI and 5 µL annexin V-FITC, (An), for 15 min at 37 °C in the dark and then analyzed using flow cytometer (annexin V-FITC Apoptosis Staining Kit, Abcam Inc., Cambridge, UK) using the florescent dye FITC signal detector and the phycoerythrin emission signal detector for annexin V-FITC binding and PI staining, respectively. Obtained data was analyzed using BD Accuri C6 Plus Software. An⁻/PI⁻ cells were viable cells. An⁺/PI⁻ and An⁺/PI⁺ cells were cells in their early and late apoptotic stage respectively. Total apoptosis was the sum of early and late apoptotic cells.