Cytotoxic Extract Screening Using AO/EB Staining

The cytotoxic extracts were assessed using the AO/EB staining technique as previously described by Ribble et al. [16 (link)]. The cells were seeded at a concentration of 100,000 cells per well in a six-well plate and incubated for 24 hours at 37°C before treatment with the active fraction LF1 at varying concentrations (5, 15 and 25 μg/ml). Following an incubation period of 24 hours, the cells were detached and pelleted. The supernatant was removed and the cells were subsequently stained with the prepared dye mixture (25 μl cold PBS and 2 μl EB/AO dye mixed in a 1:1 ratio). The stained cell suspension was transferred onto a clean glass slide and covered with a coverslip. The morphological changes relative to the untreated control was observed using the narrow blue excitation filter on an Olympus IX73 fluorescent microscope (Olympus Corporation, Shinjuku, Tokyo, JPN). The images were photographed at ×400 magnification.