Acridine orange/ethidium bromide (AO/EB) staining

This method was used to distinguish dead cells (morphological changes) from live ones. An amount of 100 μL of AO/EB mixture (100 μg/mL of each dissolved in PBS) was used for 20 minutes. After washing with PBS, the cells were observed through a fluorescence microscope (Nikon, Germany). In this staining, the AO passes through the plasma membrane of all cells and emits a green fluorescent light, but EB only passes through the membrane of damaged cells and emits a red fluorescent light. Due to the predominance of EB in relation to AO, the nuclei of live cells are integrated and green while the nuclei of primary apoptotic cells have a split chromatin and are yellow but the nucleus of cells with more advanced apoptosis show orange and fragmented chromatin.³⁰