2.9. Acridine Orange/Ethidium Bromide (AO/EB) Staining for Apoptosis

A modified method of Ribble et al. [18] for AO/EB staining of cells was applied. Cells were seeded at a density of 0.5 × 10⁵ (NHDF) or 1 × 10⁵ cells/mL (MeWo, HeLa, HepG2 and HOS) into 24-well tissue culture-treated plates, allowed to adhere overnight, then incubated with fresh complete medium (control, 0 µg CBD/mL) or various concentrations of CBD-enriched hemp oil (5, 10, 15, 20 and 25 µg CBD/mL) for 48 h. CBD-enriched hemp oil samples used for cell treatments were obtained by serial dilution of hemp oil with maximum yield of CBDA decarboxylation. After incubation, the medium was removed and cells were stained with AO/EB solution (4 µg AO/EB/mL) for 1 min, then washed with PBS and visualized in complete medium, using a DMI 3000 B inverted microscope (Leica, Wetzlar, Germany). Experiments were done in triplicate and images were acquired using the I3 filter and the 20× objective.