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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

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

Published: August 8th, 2013

DOI:

10.3791/50343

1Department of Developmental Biology, University of Pittsburgh

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.

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.

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....

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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 ch.......

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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.......

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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,.......

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The project was supported by NIH grant U01HL098180 from the National Heart, Lung, and Blood Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute or the National Institutes of Health.

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Name Company Catalog Number Comments
Name of the reagent Company Catalogue number Comments (optional)
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

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