{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,3]],"date-time":"2025-12-03T17:24:38Z","timestamp":1764782678415,"version":"3.46.0"},"reference-count":31,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2025,12,3]],"date-time":"2025-12-03T00:00:00Z","timestamp":1764720000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MPs"],"abstract":"<jats:p>A straightforward whole-mount approach has been developed that uses fluorescence imaging, mouse trachea, and a range of off-the-shelf reagents for rapidly evaluating substance toxicity within the ciliated respiratory epithelium. Using this protocol, the lumen of control trachea samples displays a typical cobblestone epithelial structure, a high density of ciliated cells, and minimal evidence of cell death, as visualized by phalloidin, acetylated tubulin, and fixable live\/dead staining, respectively. In contrast, trachea subjected to treatments that induce injury show disrupted epithelial architecture and increased cell death, indicating substance toxicity. These results support the utility of this protocol for rapidly detecting and quantifying respiratory epithelial toxicity and differential cell-type susceptibility.<\/jats:p>","DOI":"10.3390\/mps8060146","type":"journal-article","created":{"date-parts":[[2025,12,3]],"date-time":"2025-12-03T16:47:37Z","timestamp":1764780457000},"page":"146","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Optimized Whole-Mount Fluorescence Staining Protocol for Pulmonary Toxicity Evaluation Using Mouse Respiratory Epithelia"],"prefix":"10.3390","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4401-1946","authenticated-orcid":false,"given":"Richard","family":"Francis","sequence":"first","affiliation":[{"name":"Cilia Research Laboratory, College of Medicine and Dentistry, James Cook University, Townsville, QLD 4814, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"S143","DOI":"10.1513\/AnnalsATS.201802-128AW","article-title":"Airway Epithelial Differentiation and Mucociliary Clearance","volume":"15","author":"Whitsett","year":"2018","journal-title":"Ann. Am. Thorac. Soc."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"978","DOI":"10.1038\/s41385-020-00370-7","article-title":"Cellular and functional heterogeneity of the airway epithelium","volume":"14","author":"Davis","year":"2021","journal-title":"Mucosal Immunol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Button, B.M., and Button, B. (2013). Structure and function of the mucus clearance system of the lung. Cold Spring Harb. Perspect. Med., 3.","DOI":"10.1101\/cshperspect.a009720"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Wallmeier, J., Nielsen, K.G., Kuehni, C.E., Lucas, J.S., Leigh, M.W., Zariwala, M.A., and Omran, H. (2020). Motile ciliopathies. Nat. Rev. Dis. Primers, 6.","DOI":"10.1038\/s41572-020-0209-6"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Burgoyne, R.A., Fisher, A.J., and Borthwick, L.A. (2021). The Role of Epithelial Damage in the Pulmonary Immune Response. Cells, 10.","DOI":"10.3390\/cells10102763"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Carlier, F.M., de Fays, C., and Pilette, C. (2021). Epithelial Barrier Dysfunction in Chronic Respiratory Diseases. Front. Physiol., 12.","DOI":"10.3389\/fphys.2021.691227"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1038\/s41586-020-1961-1","article-title":"Tobacco smoking and somatic mutations in human bronchial epithelium","volume":"578","author":"Yoshida","year":"2020","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"L705","DOI":"10.1152\/ajplung.00492.2018","article-title":"Fourth generation e-cigarette vaping induces transient lung inflammation and gas exchange disturbances: Results from two randomized clinical trials","volume":"316","author":"Chaumont","year":"2019","journal-title":"Am. J. Physiol. Lung Cell. Mol. Physiol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Francis, R. (2023). The effects of acute hydrogen peroxide exposure on respiratory cilia motility and viability. PeerJ, 11.","DOI":"10.7717\/peerj.14899"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"382","DOI":"10.1053\/rmed.2002.1457","article-title":"Fundamentals of pulmonary drug delivery","volume":"97","author":"Groneberg","year":"2003","journal-title":"Respir. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1111\/1759-7714.13323","article-title":"Environmental pollutants damage airway epithelial cell cilia: Implications for the prevention of obstructive lung diseases","volume":"11","author":"Cao","year":"2020","journal-title":"Thorac. Cancer"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"S109","DOI":"10.1002\/ppul.27433","article-title":"Outdoor Air Pollution and Its Effects on Lung Health","volume":"60","author":"Gappa","year":"2025","journal-title":"Pediatr. Pulmonol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Bricmont, N., Bonhiver, R., Benchimol, L., Louis, B., Papon, J.F., Monseur, J., Donneau, A.F., Moermans, C., Schleich, F., and Calmes, D. (2023). Temporal Stability of Ciliary Beating Post Nasal Brushing, Modulated by Storage Temperature. Diagnostics, 13.","DOI":"10.3390\/diagnostics13182974"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3731","DOI":"10.1007\/s00405-023-07916-y","article-title":"Impact of Bepanthen((R)) and dexpanthenol on human nasal ciliary beat frequency in vitro","volume":"280","author":"Behr","year":"2023","journal-title":"Eur. Arch. Otorhinolaryngol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Fawcett, L.K., Turgutoglu, N., Allan, K.M., Belessis, Y., Widger, J., Jaffe, A., and Waters, S.A. (2023). Comparing Cytology Brushes for Optimal Human Nasal Epithelial Cell Collection: Implications for Airway Disease Diagnosis and Research. J. Pers. Med., 13.","DOI":"10.3390\/jpm13050864"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Awatade, N.T., Reid, A.T., Nichol, K.S., Budden, K.F., Veerati, P.C., Pathinayake, P.S., Grainge, C.L., Hansbro, P.M., and Wark, P.A.B. (2023). Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cells. Sci. Rep., 13.","DOI":"10.1038\/s41598-023-37828-0"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Fradique, R., Causa, E., Delahousse, C., Kotar, J., Pinte, L., Vallier, L., Vila-Gonzalez, M., and Cicuta, P. (2023). Assessing motile cilia coverage and beat frequency in mammalian in vitro cell culture tissues. R. Soc. Open Sci., 10.","DOI":"10.1098\/rsos.230185"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1183\/09031936.00153308","article-title":"Disrupted ciliated epithelium shows slower ciliary beat frequency and increased dyskinesia","volume":"34","author":"Thomas","year":"2009","journal-title":"Eur. Respir. J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"L1315","DOI":"10.1152\/ajplung.00169.2002","article-title":"Growth and differentiation of mouse tracheal epithelial cells: Selection of a proliferative population","volume":"283","author":"You","year":"2002","journal-title":"Am. J. Physiol. Lung Cell. Mol. Physiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"910","DOI":"10.1038\/nmeth817","article-title":"Fluorescence microscopy","volume":"2","author":"Lichtman","year":"2005","journal-title":"Nat. Methods"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Francis, R. (2025). A Simple Method for Imaging and Quantifying Respiratory Cilia Motility in Mouse Models. Methods Protoc., 8.","DOI":"10.3390\/mps8050113"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1007\/978-1-62703-056-4_2","article-title":"Epi-fluorescence microscopy","volume":"931","author":"Webb","year":"2013","journal-title":"Methods Mol. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Elliott, A.D. (2020). Confocal Microscopy: Principles and Modern Practices. Curr. Protoc. Cytom., 92.","DOI":"10.1002\/cpcy.68"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Rodr\u00edguez-Sevilla, P., Thompson, S.A., and Jaque, D. (2022). Multichannel Fluorescence Microscopy: Advantages of Going beyond a Single Emission. Adv. NanoBiomed Res., 2.","DOI":"10.1002\/anbr.202100084"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1042\/BIO20200075","article-title":"A beginner\u2019s guide to improving image acquisition in fluorescence microscopy","volume":"42","author":"Ogama","year":"2020","journal-title":"Biochemist"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Pawley, J.B. (2006). Handbook of Biological Confocal Microscopy, Springer. [3rd ed.].","DOI":"10.1007\/978-0-387-45524-2"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An open-source platform for biological-image analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.ab.2014.12.007","article-title":"Automatic cell counting with ImageJ","volume":"473","author":"Grishagin","year":"2015","journal-title":"Anal. Biochem."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Stansak, K.L., Baum, L.D., Ghosh, S., Thapa, P., Vanga, V., and Walters, B.J. (2024). PCP auto count: A novel Fiji\/ImageJ plug-in for automated quantification of planar cell polarity and cell counting. Front. Cell Dev. Biol., 12.","DOI":"10.3389\/fcell.2024.1394031"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Jain, A., Kim, B.R., Yu, W., Moninger, T.O., Karp, P.H., Wagner, B.A., and Welsh, M.J. (2024). Mitochondrial uncoupling proteins protect human airway epithelial ciliated cells from oxidative damage. Proc. Natl. Acad. Sci. USA, 121.","DOI":"10.1073\/pnas.2318771121"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"722","DOI":"10.1111\/resp.12542","article-title":"Bronchial epithelial cells: The key effector cells in the pathogenesis of chronic obstructive pulmonary disease?","volume":"20","author":"Gao","year":"2015","journal-title":"Respirology"}],"container-title":["Methods and Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2409-9279\/8\/6\/146\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,3]],"date-time":"2025-12-03T17:19:51Z","timestamp":1764782391000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2409-9279\/8\/6\/146"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,3]]},"references-count":31,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["mps8060146"],"URL":"https:\/\/doi.org\/10.3390\/mps8060146","relation":{},"ISSN":["2409-9279"],"issn-type":[{"value":"2409-9279","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,3]]}}}