Gkt137831

The rate of hydrogen peroxide produced by HPASMC and HPAEC was measured in cells seeded at density 40000 cells per well in 96-well plate using the Amplex® Red Hydrogen Peroxide Assay Kit (Thermo Fisher Scientific, Burlington, ON, Canada, cat# A22188) as published^{24 (link)}. Briefly, the cells were washed twice in Hanks’ Balanced Salt Solution (HBSS, Thermo Fisher Scientific, Burlington, ON, Canada, cat# 14025076) and 50 µM of Amplex® Red reagent and 0.1 U/mL of horseradish peroxidase were added. The total volume was adjusted to 100 µL with the provided reaction buffer, and the fluorescence was measured at excitation 540 nm and emission 590 nm using the plate reader (Synergy H1, Biotek, Winooski, VT). The kinetics of the reaction was recorded every 2 minutes for 60 minutes. The rate of H₂O₂ production was evaluated by subtracting the final fluorescence intensity from the initial fluorescence intensity, divided by time. The data were presented as a relative rate of peroxide generated per minute.
To evaluate the role of NADPH oxidases (NOX) enzymes in hydrogen peroxide production and HMGB1 dimerization, the cells were pre-treated with 25 nM of NOX1/NOX4 inhibitor, GKT137831 (Medkoo Biosciences, Morrisville, NC, cat # 522357) for 3 hours before measuring the H₂O₂ production.