Ix71sif 3

The viability of cells was assessed by measuring their ability to metabolize 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) (as described in [5 (link)]) and by a nuclear fluorescent staining assay. In the first experimental series, C6 cells were treated with different concentrations of either plumbagin, menadione, or lawsone for 24 h. Concentrations for plumbagin and menadione were 1 µM, 3 µM, 5 µM, 8 µM, 10 µM, 12.5 µM, 15 µM, 20 µM, and 25 µM and for lawsone were 10 µM, 25 µM, 50 µM, 75 µM, 100 µM, 250 µM, 500 µM, 750 µM, and 1000 µM. In experiments with ascorbate, C6 cells were treated with 5 µM and 15 µM plumbagin, together with 100 µM and 250 µM of ascorbic acid for 24 h. After incubation with 1, 4-naphthoquinones, the cells were double-stained with Hoechst 33,342 (15 µg/mL) and propidium iodide (PI; 5 µg/mL) for 15 min, and the viability was assessed under the fluorescence microscope OLYMPUS IX71SIF-3 (Olympus Corporation, Tokyo, Japan). IC₅₀ was calculated by SigmaPlot 13.0 version (Systat Software Inc., San Jose, CA, USA) software by means of the four-parameter logistic function.

To investigate the effect of extracts on cell viability in oxidative stress conditions cells were treated: (1) high oxidative stress conditions (100 µM H₂O₂ for 24 h with different amounts of investigated extracts) or (2) moderate oxidative stress conditions (75 µM H₂O₂ for 6 h with different amounts of investigated extracts). After the treatments, viability staining of nuclei was performed by adding propidium iodide (PI, 3 µg/mL) and Hoechst 33,342 (6 µg/mL) to the incubation media and allowing the dyes to penetrate cells, incubating for 5 min, at 37 °C. The stained cells in the cultures were visualized under a fluorescent microscope OLYMPUS IX71SIF-3 (Olympus Corporation, Tokyo, Japan). Hoechst33342-only-positive nuclei exhibiting blue fluorescence were considered viable, and Hoechst3334-plus-PI-positive nuclei stained magenta were identified as necrotic. The image analysis was performed using ImageJ software.