HepG2 cells were incubated in the presence or absence of NFOH or BZL for 24 h, as above. For the quantitation of reactive oxygen species (ROS), 5 µM CellROX Green reagent was added to each well in complete media, and cells were incubated at 37°C for 30 minutes. CellROX Green is cell-permeant and non-fluorescent, or very weakly fluorescent, in the reduced state. Upon oxidation, the reagents exhibit strong fluorescence and remain localized within the cell. Cells were washed with PBS and analyzed by flow cytometry.
For assessing the effect of NFOH and BZL in inducing DNA damage, HepG2 cells were treated with NFOH or BZL for 24 h, as above. Cells were harvested, washed with PBS, fixed with 3.7% paraformaldehyde for 15 min at 4°C, and permeabilized with 90% methanol. Cells were then incubated at room temperature for 2 h with mouse anti-8-oxo-dG antibody (250-fold dilution, EMD Milipore, Billerica, MA) and for 30 min with PE-conjugated, anti-mouse IgG (eBioscience, San Diego, CA). Cells stained with isotype-matched IgGs were used as controls. Samples were visualized on an LSRII Fortessa Cell Analyzer, acquiring 30–50,000 events in a live cell gate, and further analysis performed by using FlowJo software (version 7.6.5, Tree-Star, San Carlo, CA).
For assessing the effect of NFOH and BZL in inducing DNA damage, HepG2 cells were treated with NFOH or BZL for 24 h, as above. Cells were harvested, washed with PBS, fixed with 3.7% paraformaldehyde for 15 min at 4°C, and permeabilized with 90% methanol. Cells were then incubated at room temperature for 2 h with mouse anti-8-oxo-dG antibody (250-fold dilution, EMD Milipore, Billerica, MA) and for 30 min with PE-conjugated, anti-mouse IgG (eBioscience, San Diego, CA). Cells stained with isotype-matched IgGs were used as controls. Samples were visualized on an LSRII Fortessa Cell Analyzer, acquiring 30–50,000 events in a live cell gate, and further analysis performed by using FlowJo software (version 7.6.5, Tree-Star, San Carlo, CA).