Mitochondrial morphology and mitochondrial reactive oxygen species assays using fluorescence microscopy

PAECs were seeded on a coverslip in a 24-well plate. The cells were treated with vehicle or 50 µM of adenosine plus 50 µM of DCF for up to 48 h, a treatment that we have previously shown to increase EC adenosine levels.^32,35 The cells were washed with the culture media twice followed by incubation with MitoTracker Green to stain mitochondria in the media for 20 min at 37 ℃. The media were removed, and the cells were washed twice with fresh media. The cells were then co-stained with MitoSOX Red to label mitochondrial reactive oxygen species (ROS) in the culture media for 20 min at 37 ℃. After washing twice with culture media, the coverslips were mounted onto microscope slides with a drop of warm media and sealed with nail polish. Images were captured by a fluorescence microscope. For the experiments where MitoTEMPO was used, the PAECs were pre-treated with 5 µM of MitoTEMPO or vehicle for 1 h. After pretreatment, the cells were treated with adenosine plus DCF (AD) in the presence or absence of MitoTEMPO for 30 h. The cells were then stained with MitoTracker green and processed for fluorescent microscopy.