2.6. Quantification of H2O2 and NOX4 activity

Cellular production of H₂O₂ was evaluated by its fluorescent reaction product with coumarin boronic acid (Cayman, 14051) in the presence of l-NAME (Cayman, 80210) and taurine (Cayman, 27031) [[31], [32], [33]]. Coumarin boronic acid (CBA) is a sensitive probe for hydrogen peroxide, but necessary control must be included to rule out interference from peroxynitrite and hypochlorous acid. l-NAME inhibits NOS activity and therefore prevents peroxynitrite production, while taurine scavenges hypochlorous acid. Therefore, both l-NAME and taurine are added to the assay buffer to improve the probe's specificity [34]. For endothelial cells, cells were plated and transfected in a 96-well plate. To measure H₂O₂, the medium was changed to the assay buffer (20 mM HEPES pH 7.5, 10 μM EDTA, 100 μM l-NAME, 1 mM taurine, and 0.01% bovine serum albumin in DMEM). For each group, negative controls were included by adding catalase to the final concentration of 500 μg/ml. Reactions were started by adding 20 μM CBA probe. Fluorescence (350/450 nm) was measured kinetically at 37 °C for 4 h. At the end of the assay, endothelial cells were stained with crystal violet to determine the number of cells. To calculate the reaction rate, the log phase slope was normalized with the crystal violet staining results. For HEK293(FT) cells, since they did not withstand repeated rinsing during crystal violet staining, a different strategy was used for normalization. After transfection, HEK293(FT) cells were dislodged, counted, and resuspended in the assay buffer so that each well in a 96-well plate contains 120,000 cells at the time of the assay. Otherwise, the CBA assay was performed the same as for HAECs. Additionally, cells were pelleted from the remaining cell suspension for protein quantification to normalize the result. Importantly, for each treatment, the fluorescent signal from a corresponding catalase control was included, and the catalase-inhibitable amount was designated as H₂O₂. The CBA probe is membrane permeable, while catalase is not. Therefore, the catalase-inhibitable signal represents extracellular H₂O₂ concentration, which correlates with its intracellular level.