Cytotoxicity was assessed on HEK293 cells [66 (link)]. The methodology followed was described previously [67 (link)]. Cytotoxicity was determined for of the combination silver acetate/plazomicin at various concentrations. One thousand cells per well were cultured on flat-bottom 96-well, black microtiter plates. After 12 h incubation, the testing compounds were added at the desired concentrations and incubation was continued for 24 h. Following, the cells were washed with sterile D-PBS, resuspended in the LIVE/DEAD reagent (2 μM ethidium homodimer 1 and 1 μM calcein-AM) (Molecular Probes), and incubated for 30 min at 37 °C. At this moment the fluorescence levels corresponding to dead and live cells (645 nm and 530 nm, respectively) were measured. The percentage of dead cells was calculated relative to the untreated cells. Maximum toxicity was calculated treating cells with 70% methanol for 20 min. Experiments were conducted in triplicate. The results were expressed as mean ± SD of three independent experiments. HEK293 cells were purchased from BEI resources (Manassas, VA, USA), catalog number NR-9313.
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