<em>Ex vivo</em> Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

Abstract

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/ sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy. Video LinkThe video component of this article can be found at

Assessment of ciliary phenotype in primary ciliary dyskinesia by micro-optical coherence tomography

“…HSVM imaging. For HSVM imaging, tracheal samples were removed and dissected as described above and as previously reported (34). Tracheal samples were then transferred to a glass-bottom culture dish with L-15 media and covered with a housing composed of hollowed silicone gel and topped with a glass coverslip as previously described (34).…”

“…For HSVM imaging, tracheal samples were removed and dissected as described above and as previously reported (34). Tracheal samples were then transferred to a glass-bottom culture dish with L-15 media and covered with a housing composed of hollowed silicone gel and topped with a glass coverslip as previously described (34). Cilia HSVM imaging was carried out using DIC imaging on a Leica DMRE inverted microscope with a ×100 oil objective (NA: 1.40) and a Phantom v4.2 camera (Leica) operated at 200 frames per second.…”

Cilia and Mucociliary Clearance

“…Other investigators studying mouse models have recorded the movement of fluorescent particles along the trachea or nasopharynx of the mouse (e.g., Ostrowski et al 2010) or measured the recovery of fluorescent beads from the lungs after a specified time (Look et al 2001; Grubb et al 2016). Alternatively, investigators have also used ex vivo preparations of trachea or bronchi to investigate MCC (Cooper et al 2013; Francis and Lo, 2013). Although these studies provide valuable information, the techniques are invasive and the system is not easy to manipulate.…”

CCDC151 Mutations Cause Primary Ciliary Dyskinesia by Disruption of the Outer Dynein Arm Docking Complex Formation

“…19,42 TEM in mice and zebrafish was prepared as previously reported, 11 and video microscopy was performed as previously reported. 44,[47][48][49] cDNA Cloning CCDC114 and CCDC151 were cloned by nested PCR from human bronchial epithelial cell cDNA (ScienCell, cat no. 3214) using KOD polymerase according to manufacturer's directions and recombined with pDONR201 Gateway vector via BP Clonase II reaction.…”