Ros glotm h2o2 assay kit

Measurement of hydrogen peroxide was carried out with the ROS-Glo^TMH₂O₂ Assay kit (Promega, Madison, WI), according to the manufacturer’s instructions. Briefly, 80 μl of serum from adult control and ATZ-treated mice were mixed with 20 μl of H₂O₂ substrate and held for 6 h at 37 °C. Then, 100 μl of ROS-Glo^TM detection solution was added and incubated for 20 min at room temperature, and relative luminescence was measured in a plate reader.

The level of ROS, H₂O₂ was measured by the ROS-GloTM H₂O₂ Assay kit (Promega). For in vitro experiment, 5000 cells were seeded in a 96-well white plate and incubated in normal culture conditions for 24 h. The cells were treated with H₂O₂ for 18 h under normal culture conditions. To perform the assay, the cells were incubated with 25 µl of H₂O₂ substrate solution each well for 6 hours. Then 100 µl of ROS-Glo^™ Detection Solution was applied into each well and incubated at room temperature for 20 min. The luminescent signal was quantified by a luminescence plate reader (Bio-Rad). For human serum samples, each 100 µl of serum sample was incubated with 25 µl of H₂O₂ substrate solution for 6 h and followed by the same step as done in in vitro experiment. Each experiment was repeated three times.

The hydrogen peroxide (H₂O₂) luminescence detection assay was performed using the ROS-GloTM H₂O₂ Assay Kit (Promega, Madison, WI, USA). To perform the experiment, in a 96-well plate, 1 × 105 SCC9 cells were seeded in DMEM F12 with 10% FBS medium per well. The cells were incubated for 24 h and, after this period, the medium was removed and the cells were treated with 2 × IC50 from the 09.07 and 14.05 fractions or with the DMSO control at 6, 24, and 48 h. Cell-free wells were used as a control. Menadione (M9529-Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control. After the indicated time, the H₂O₂ substrate was used for all treatments. After that, the detection solution was added and the plates were read in the luminometer (Turner Designs, TD 20/20, San Jose, CA, USA).

Immortalized porcine bronchial epithelial cells were seeded in a 96-well plate at 80% confluence the night before the experiment. The cells were incubated with 20 μg of purified rNFOR, 80 μL of three strains of Mhp diluted in a final volume of DMEM/F12 plus 2% FBS culture medium, or 80 μL PBS. Then, 20 μL of H₂O₂ Substrate solution from the ROS-Glo^TM H₂O₂ Assay kit (Cat No. G8820, Promega, Madison, WI, United States) was added to the cells and mixed. Thus, the final well volume was 100 μL, and the final H₂O₂ Substrate concentration was 25 μM. The plate was incubated in a 37°C incubator for 6 h prior to adding 100 μL of ROS-Glo Detection Solution and incubated at RT for 20 min. The relative luminescence units (RLUs) were recorded using a plate reader. PBS was used as a negative control, and positive controls were the RLUs from three Mhp strains (strain JS, J, and 168 L, 1 × 10⁸ CCU/mL). Detection of reactive oxygen species (ROS) levels in the cells incubated with rNFOR, three Mhp strains or PBS was performed in five independent replicates.