2.5. Detection of apoptotic morphological changes by acridine orange and ethidium bromide (Ao/EtBr) staining

D-pinitol stimulated apoptotic morphological alterations were studied in human leukemia MOLT-4 cells by employing AO/EtBr dual staining followed by the earlier methods Aithal et al., (2009). Briefly, from the parental flask, cells were collected by trypsinization and 1 × 10⁶ cells per well were loaded in 6 well plates. Then, cells were supplemented with D-pinitol for 24 h. After that cells were thoroughly washed thrice with PBS at 5 min interval. Then, 1 μl of AO/EtBr (5 mg/ml concentration) was amalgamated and incubated under dark for 30 min at 37 °C. Excessive dyes were removed by repeated washing with PBS. Finally, cells were inspected under the fluorescence microscope and images were taken.