Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

Summary

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

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.

Introduction

Analysis of motile cilia function in the airway epithelia is experimentally important for elucidating the genetic and environmental factors that can affect mucociliary clearance and pulmonary health ¹. The simple protocol developed for imaging the mouse airway epithelia provides an efficient method to interrogate airway cilia motility in mutant and knockout mouse models and require only basic skills in mouse tracheal tissue dissection and ex vivo imaging of airway cilia motility with high resolution videomicroscopy. This protocol was established and refined during a large-scale mouse mutagenesis screen to allow rapid evaluation of motile cilia func....

Protocol

1. Reagents Setup

1.1 Dissection and Imaging Medium

Leibovitz's L-15 medium (L15) is supplemented with FBS (10%) and Penicillin-Streptomycin (100 units/ml of penicillin G sodium and 100 μg/ml of streptomycin sulfate) is used during both harvest and imaging of trachea samples.

2. Shallow Walled Culture Chamber Assembly

The chamber used to hold trachea tissue is shown in Figure 1. The floor of the chamber is the glass bottomed 35 mm culture dish. The top and side of the chamber is generated by placing a small piece of 0.3 mm thick silicone sheetin....

Results

Control airway cilia should be clearly visible and seen to beat in a coordinated manner (Supplemental Movie 1; movie playback is slowed to 15% real time), with noticeable flow in the direction of cilia beat (Supplemental Movie 2; movie playback is 100% real time). Quantitation of cilia movies should yield results similar to that seen in Figure 3. Collection of high-speed DIC movies makes possible the quantification of cilia beat frequency (Figure 3C) and.......

Discussion

Measurement of cilia beat frequency (CBF) is relatively easy using high power microscope objectives and fast image acquisition hardware ^13,15, and explains why CBF measurements form the basis of most studies investigating mucociliary clearance during health and disease. However, while CBF measurement is essential for understanding mucociliary clearance, measurement of CBF alone ignores the underlying importance of both ciliary generated flow and cilia beat waveform, both of which are more difficult to measure,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Leibovitz’s L-15 Medium	Invitrogen	21083-027	No phenol red
Fetal bovine serum	Hyclone	SH30088.03
Penicillin-Streptomycin	Invitrogen	15140-122
2x fine forceps	Roboz	RS-4976
Dissection scissors	Roboz	RS-5676
Micro dissection scissors	Roboz	RS-5620
Scalpel	Roboz	RS-9801-15
P1000 pipetman	Gilson, Inc	F123602
P1000 tips	Molecular BioProducts	2079E
18 mm round glass cover slips	Fisher Scientific	430588
Plastic 35 mm culture dishes	Corning	430588
Glass bottom 35 mm culture dishes	Warner Instruments	W3 64-0758
Silicone sheet 0.012" (0.3 mm) thick	AAA Acme Rubber Co	CASS-.012X36-63908
0.20 μm diameter Fluoresbrite YG Carboxylate Microspheres	Polysciences	09834-10
Inverted microscope, with 100x oil objective and DIC filters	Lecia	DMIRE2	Brand is not critical.
100-watt mercury lamp, epifluorescent FITC excitation/emission filters	Lecia		Brand is not critical.
Microscope stage Incubator	Lecia	11521749	Not required if imaging cilia at room temperature
High-speed camera bright field	Vision Research	Phantom v4.2	Brand is not critical. Must be faster than 125 fps
High-speed fluorescent camera	Hamamatsu	C9100-12	Brand is not critical. Must be faster than 10 fps
Movie analysis software	National Institutes of Health	ImageJ with MtrackJ plugin