Determination of Morphological Features of Apoptosis

A total of 5 × 10⁵ cells were plated on 60-mm dishes, allowed to attach overnight, and then treated with PL (15 μM) in the presence or absence of NAC (5 mM) for 2 h. Twenty-four hours later, the cells were fixed, washed twice with PBS, and stained with Hoechst or PI or acridine orange and ethidium bromide staining solution according to the manufacturer’s instructions. The cells were observed and imaged using a fluorescent microscope (Nikon, Tokyo, Japan) with 20× amplification.