Imaging and Quantification of Nitric Oxide and Ciliary Beating

Imaging of NO and CBF was as described [28 (link)]. For NO, ALI cultures were loaded with DAF-FM by incubation in 10 μM DAF-FM diacetate (ThermoFisher) on the apical side in HBSS plus 5 μM carboxy-PTIO (to scavenge baseline NO; Cayman Chemical) for 90 min. For submerged CFBEs on 8-well chambered coverglass (CellVis, Sunnyvale, CA, USA), loading was the same but for 45 min instead of 90 min. After washing, imaging was performed using an IX-83 microscope (10x 0.4 NA PlanApo objective, Olympus Life Sciences, Tokyo, Japan) with LED illumination (Excelitas Technologies LED120Boost), 16-bit Orca Flash 4.0 sCMOS camera (Hamamatsu, Japan), standard FITC filter set (470/40 nm excitation, 495 lp dichroic, and 525/40 nm emission; 49002-ET, Chroma Technologies) and MetaFluor (Molecular Devices, Sunnyvale, CA, USA). For cilia beating, cultures were maintained at ~28 °C in DPBS (+1.8 mM calcium) on the apical side and HEPES-buffered HBSS supplemented with 1× MEM amino acids (Gibco) on the basolateral side. Sisson-Ammons Video Analysis software was used to measure whole-field CBF. Diphenhydramine and all other reagents used were from Millipore Sigma unless otherwise specified.