HSNE cultures were visualized on an inverted microscope (Fisher Scientific, Pittsburgh, PA) using a 20× objective lens. A high-speed monochromatic digital video camera (Model A602f-2; Basler AG, Ahrensburg, Germany) was used to record data at a sampling rate of 100 frames/s. Images were analyzed with Sisson-Ammons Video Analysis (SAVA, version 2.0.8 W) system.5 (link),32 (link),33 ,35 (link)–37 (link) Using the inverted microscope, large regions of beating cilia were identified within HSNE ALI cultures for each experiment. Captured images were then analyzed for CBF with virtual instrumentation software. Prior to apical fluid application to cell monolayers (addition of apical fluid stimulates CBF), baseline recordings of CBF were performed in both control and hypoxia-exposed cultures for 5 minutes. After baseline CBF measurement, a solution consisting of 100 μL of phosphate-buffered saline (PBS) and 20 μmol/L forskolin was added and stimulated CBF was recorded for another 15 minutes and compared with vehicle control.
Reported CBFs represent the arithmetic means of all data points. All experiments were carried out at room temperature (23°C).
Reported CBFs represent the arithmetic means of all data points. All experiments were carried out at room temperature (23°C).
