Quantifying Cellular Reactive Oxygen Levels

The membrane-permeable probes HE and DCFH-DA were used to assess levels of ROS using methods that have been described previously [28 (link), 29 (link)]. For the in vitro study, HBECs were incubated in culture medium containing 10 μM HE at 37°C for 30 min. After stimulation with CSE for the desired time, the culture medium was removed for the measurement of extracellular ROS levels. The cells were washed and detached with trypsin/EDTA to allow the measurement of intracellular ROS levels. For the in vivo study, the supernatant of the first BALF sampled from all study groups was incubated with 10 μM DCFH-DA at 37°C for 15 min. The fluorescence intensities of the culture medium, cells, and BALF samples were then analyzed using a multilabel counter (PerkinElmer, Waltham, MA, USA). Images of the cells were also obtained by examining them using a Nikon TE2000-U florescence microscope (Tokyo, Japan).