2.4. Amplex red hydrogen peroxide assay

Hydrogen peroxide levels were measured following C-PAC treatment of EAC and BE cells using the Amplex Red Hydrogen Peroxide/Peroxidase kit according to the manufacturer’s recommendations (ThermoFisher Scientific, Waltham, MA). JHAD1 and OE19 EAC cells were plated at 10E3 and 15E3 cells/well, respectively in 96-well black walled, clear bottom plates (Greiner Bio One, Monroe, NC). CP-C BE cells were plated at 8E3 cells/well in the same plates. Cells adhered for 26–30 h, washed once with PBS and treated for 6 h with vehicle, C-PAC [50–100 μg/mL], the inducer 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ; 20 μM final concentration; Sigma Aldrich, St. Louis, MO) and the inhibitor diethylene triamine pentaacetic acid (DTPA; 100 μM final concentration; Sigma Aldrich, St. Louis, MO). After 6 h, the supernatant was transferred to a new plate and the cells were washed. The cells, the supernatant from the cells and the reaction-no cells in medium were assayed independently for hydrogen peroxide 30 min after addition of the Amplex Red substrate. Fluorescence was measured using the SpectraMax^® i3 with excitation/emission wavelengths of 545–590 nm. A minimum of 4 wells were analyzed for each treatment and the data expressed as mean relative fluorescence units ± SEM.