Neutral Red Cytotoxicity Assay

Cell cytotoxicity was also analyzed using a neutral red dye (Sigma Aldrich, St. Louis, MO, USA) according to the methodology previously described by Zagórska-Dziok et al. [57 (link)]. As in the case of the AB assay, both cell types were placed in 96-well plates and cultured for 24 hours. After the medium was aspirated, the cells were subjected to a 24-hour exposure to the tested samples at concentrations of 100, 250 and 1000 μg/mL. The negative control was DMEM medium without the addition of elderberry extract or ferment. After this time, the solutions of the tested samples were aspirated from the wells, and the cells were subjected to a 2-hour incubation with a solution of a neutral red dye (40 μg/mL). The cells were then washed with phosphate-buffered saline (PBS), and then a decolorizing solution (EtOH/AcCOOH/H₂O₂, 50%/1%/49%) was added. The prepared plates were shaken for 10 minutes, and then the uptake of the neutral red dye by the cells was measured by determining the optical density (OD) of the eluted dye at 540 nm in a FilterMax F5 microplate-reader spectrophotometer (Thermo Fisher). Three independent experiments were performed, during which each extract and ferment concentration was tested in triplicate. The results are presented as the percentage of the amount of dye retained compared with the control cells, for which a value of 100% was assumed.