Acridine Orange/Propidium Iodide Staining of Treated Cells

To assess the morphological pattern of cell death induced by mono-RL and Piceatannol treated cells an acridine orange (AO)/propidium iodine (PI) staining technique was utilized [39 (link)]. HCT 116, Caco2 and CCD-841-CoN cells were cultured and treated as previously described in Section 2.7. Following treatment, cells were washed three times with sterile PBS (Thermo Fisher Scientific) and subsequently incubated for 3 min with 100 μg mL⁻¹ AO and PI (Merck Life Science, Bengaluru, India) mixed at a ratio of 1:1. Cells were then re-washed with sterile PBS (Thermo Fisher Scientific) and stained cells were imaged at 200× magnification using an Eclipse TS100 fluorescence microscope (Nikon). The excitation and emission wavelengths for AO were 493 nm and 535 nm, and for PI were 535 nm and 614 nm. All experiments were carried out in triplicate with three technical replicates per treatment condition. Each replicate was imaged three times and images for publication were randomly selected for publication using a computer. Scale bars (100 μm) were added to images using ImageJ 1.53t software [38 (link)].