ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We thank Dr. Mark T. Gladwin (Dean and Professor, University of Maryland School of Medicine) for guidance on study design. We also thank the University of Maryland School of Medicine&#8217;s Confocal Microscopy Core (Baltimore, MD) for access to confocal microscopy resources. This work was supported by National Institutes of Health (NIH) grant 5R01HL168775 to Dr. Paola Corti.

<ol>
	<li>Alc&#225;zar&#8211;Navarrete, B., Ruiz Rodr&#237;guez, O., Conde Baena, P, Romero Palacios, P. J., Agusti, A. Persistently elevated exhaled nitric oxide fraction is associated with increased risk of exacerbation in COPD. Eur Respir J. 2018;51(1):1701457.</li>
	<li>Krantz, C., Janson, C., Alving, K., Malinovschi, A. Nasal nitric oxide in relation to asthma characteristics in a longitudinal asthma cohort study. Nitric Oxide 2021;106:1&#8211;8.</li>
	<li>Thomas, S. R., Kharitonov, S. A., Scott, S. F., Hodson, M. E., Barnes, P. J. Nasal and exhaled nitric oxide is reduced in adult patients with cystic fibrosis and does not correlate with cystic fibrosis genotype. Chest 2000;117(4):1085&#8211;9.</li>
	<li>Shapiro, A. J. et al. Nasal nitric oxide measurement in primary ciliary dyskinesia. A technical paper on standardized testing protocols. Ann Am Thorac Soc. 2020;17(2):e1&#8211;e12 .</li>
	<li>Legendre, M., Zaragosi, L. E., Mitchison, H. M. Motile cilia and airway disease. Semin Cell Dev Biol. 2021;110:19&#8211;33.</li>
	<li>Li, D., Shirakami, G., Zhan, X., Johns, R. A. Regulation of ciliary beat frequency by the nitric oxide&#8211;cyclic guanosine monophosphate signaling pathway in rat airway epithelial cells. Am J Respir Cell Mol Biol. 2000;23(2):175&#8211;81.</li>
	<li>Rochon, E. R. et al. Cytoglobin regulates NO-dependent cilia motility and organ laterality during development. Nat Commun. 2023;14(1): 8333.</li>
	<li>Mikhailik, A. et al. nNOS regulates ciliated cell polarity, ciliary beat frequency, and directional flow in mouse trachea. Life Sci Alliance 2021;4(5):e202000981.</li>
	<li>Schamberger, A. C., Staab&#8211;Weijnitz, C. A., Mise&#8211;Racek, N., Eickelberg, O. Cigarette smoke alters primary human bronchial epithelial cell differentiation at the air&#8211;liquid interface. Sci Rep. 2015;5:8163.</li>
	<li>Gindele, J. A. et al. Intermittent exposure to whole cigarette smoke alters the differentiation of primary small airway epithelial cells in the air&#8211;liquid interface culture. Sci Rep. 2020;10(1):6257.</li>
	<li>Ghosh, B. et al. Effect of sub-chronic exposure to cigarette smoke, electronic cigarette and waterpipe on human lung epithelial barrier function. BMC Pulm Med. 2020;20(1):216.</li>
	<li>Baldassi, D., Gabold, B., Merkel, O. Air-liquid interface cultures of the healthy and diseased human respiratory tract: promises, challenges and future directions. Adv Nanobiomed Res. 2021;1(6):2000111.</li>
	<li>Bessa, M. J. et al.&#160;In vitro&#160;toxicity of industrially relevant engineered nanoparticles in human alveolar epithelial cells: Air-liquid interface&#160;versus&#160;submerged cultures. Nanomaterials (Basel) 2021;11(12):3225.</li>
	<li>Allouche, Y. et al. Comparison of submerged and air liquid interface exposure: limitations and differences in the toxicological effects evaluated in bronchial epithelial cells. Environ Res. 2025;279(Pt 2):121856.</li>
	<li>Li, J., LoBue, A., Heuser, S. K., Leo, F., Cortese-Krott, M. M. Using diaminofluoresceins (DAFs) in nitric oxide research. Nitric Oxide. 2021;115:44&#8211;54.</li>
	<li>Jiang S. et al. Real&#8211;time electrical detection of nitric oxide in biological systems with sub&#8211;nanomolar sensitivity. Nat Commun. 2013;4:2225.&#160;</li>
	<li>Brown M. D, Schoenfisch M. H. Electrochemical nitric oxide sensors: Principles of design and characterization. Chem Rev. 2019;119(22):11551&#8211;75.</li>
	<li>Corti, P. et al. Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells. Proc Natl Acad Sci U S A 2016;113(30):8538&#8211;43.</li>
	<li>Zhao, X. J. et al. Mechanisms for cellular NO oxidation and nitrite formation in lung epithelial cells. Free Radic Biol Med. 2013;61:428&#8211;37.</li>
	<li>Lundberg, J. O. Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov. 2008;7(2):156&#8211;67.</li>
	<li>Rochon, E. R., Corti, P. Globins and nitric oxide homeostasis in fish embryonic development. Mar Genomics 2020;49:100721.</li>
	<li>Giordano, D., Verde, C., Corti, P. Nitric oxide production and regulation in the teleost cardiovascular system. Antioxidants (Basel) 2022;11(5):957.</li>
	<li>MacArthur, P. H., Shiva, S., Gladwin, M. T. Measurement of circulating nitrite and S&#8211;nitrosothiols by reductive chemiluminescence. J Chromatogr B Analyt Technol Biomed Life Sci. 2007;851(1&#8211;2):93&#8211;105.</li>
	<li>Basu, S., Ricart, K., Gladwin, M. T., Patel, R. P, Kim-Shapiro, D. B. Tri-iodide and vanadium chloride based chemiluminescent methods for quantification of nitrogen oxides. Nitric Oxide 2022;121: 11&#8211;19.</li>
	<li>Brody, S. L., Yan, X. H., Wuerffel, M. K., Song, S. K., Shapiro, S. D. Ciliogenesis and left-right axis defects in forkhead factor HFH-4-null mice. Am J Respir Cell Mol Biol. 2000;23(1): 45&#8211;51.</li>
	<li>Jain, R. et al. Temporal relationship between primary and motile ciliogenesis in airway epithelial cells. Am J Respir Cell Mol Biol. 2010;43(6): 731&#8211;9.</li>
	<li>Eenjes, E. et al. A novel method for expansion and differentiation of mouse tracheal epithelial cells in culture. Sci Rep. 2018;8(1): 7349.</li>
	<li>You, Y., Richer, E. J., Huang, T., Brody, S. L. Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population. Am J Physiol Lung Cell Mol Physiol. 2002;283(6): L1315&#8211;21.</li>
	<li>Pfeiffer, S., Leopold, E., Schmidt, K., Brunner, F., Mayer, B. Inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME): requirement for bioactivation to the free acid, NG-nitro-L-arginine. Br J Pharmacol 1996;118(6):1433&#8211;40.&#160;</li>
	<li>Rochon, E. R., Corti, P., Gladwin, M. T. Hemoglobin &#945; in Pulmonary Endothelium: Ironing Out Nitric Oxide Signaling. Am J Respir Cell Mol Biol. 2017;57(6):639&#8211;41.</li>
	<li>van Faassen, E. E. et al. Nitrite as regulator of hypoxic signaling in mammalian physiology. Med Res Rev. 2009;29(5):683&#8211;741.&#160;</li>
</ol>

The authors declare no conflicts of interest.

Multi&#8211;ciliated cells within the airway epithelium are critical for NO synthesis and signaling, which in turn is essential for regulating ciliary beating, mucociliary clearance, and airway homeostasis6,7,8. NO is a highly reactive molecule and even small changes in NO levels are associated with airway diseases1,2,3,<sup ...

A hallmark of several airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and primary ciliary dyskinesia (PCD), is altered nitric oxide (NO) homeostasis measurable as changes in exhaled NO levels, which can largely vary in these diseases, being elevated in COPD and asthma1,2, while drastically reduced in CF and PCD3,4. These diseases commonly exhibit abnormalities in airway epithelial motile cilia, the key effectors of mucociliary clearance5. Because NO and its downstream signaling pathways are critical regulators of ciliary motility, understanding how NO production and signaling are altered in these contexts is essential for elucidating mechanisms of mucociliary dysfunction. Accordingly, a method that enables precise detection of NO production and release in airway epithelia provides a valuable tool for dissecting the regulation and physiological role of this signaling molecule. Such approaches are particularly useful in experimental settings where direct measurement of NO is challenging due to its short half-life and rapid reactivity.
NO regulates airway cilia biogenesis and function by influencing ciliated cell polarity and the coordinated beating of motile cilia6,7,8. However, the mechanisms that maintain NO homeostasis in the airways remain incompletely understood. Airway epithelial cultures differentiated at air&#8211;liquid interface (ALI) provide a valuable in vitro system for investigating these mechanisms. At present, this model is widely used to study the impact of environmental insults on the airway epithelium9,10,11 and to elucidate the pathophysiology of airway diseases12. Compared with conventional submerged cultures13,14, ALI&#8211;differentiated epithelia more faithfully reproduce the architecture and function of the native airway, including epithelial polarization, mucociliary differentiation, and realistic gas&#8211;liquid exchange, making them especially suitable to study apical NO release, signaling, and related ciliary function. This system is therefore well-suited for experiments aimed at quantifying NO-derived metabolites released into the airway lumen, particularly under conditions of epithelial differentiation or pharmacological manipulation.
Accurate measurement of NO concentration is essential, as both excessive and deficient NO levels can impair ciliary function. Currently, fluorescent probe&#8211;based methods (e.g., diaminofluoresceins, DAFs) available for detecting NO are only semi-quantitative and are limited by pH sensitivity, oxygen dependence and lack of specificity, as they can react with cellular oxidants and antioxidants, resulting in semi-quantitative measurements15. In addition, electrochemical sensors (e.g., NO-sensitive amperometric electrodes) enable real&#8211;time detection of NO16 but can be difficult to implement in complex biological systems due to NO short half&#8211;life, interference from redox-active species biofouling of the electrode surface, and challenges in maintaining stable calibration17. Alternative chemiluminescence-based approaches including direct gas-phase NO detection and reductive assays targeting different nitrogen oxide species, have also been described, but their application to airway epithelial cultures remains unexplored. These limitations highlight the need for sensitive, quantitative methods that can reliably detect low levels of NO-derived species in small-volume biological samples. To overcome these limitations, we established a protocol to quantify NO in apical secretions collected from ALI-differentiated airway epithelia using a Nitric Oxide Analyzer (NOA). The NOA is a highly sensitive chemiluminescence&#8211;based detector traditionally used to measure NO metabolites in chemical samples and more recently adapted for the analysis of biological specimens, including blood, tissue homogenates, and cell culture supernatants7,18,19. NO is a highly reactive signaling molecule rapidly oxidized to nitrite in tissues under conditions of normal oxygen levels, with an exceptionally short half-life. Thus, nitrite as end product of NO oxidation serves as an indicator of total NO production in biological systems20,21,22. This sensitive and reproducible chemiluminescence method allows detection of nitrite/NO at picomolar levels23,24, making it ideal for measuring small changes in low&#8211;volume biological samples such as those derived from MTEC ALI cultures. However, this approach is best suited for conditions where nitrite accumulation reflects recent NO production and may be influenced by factors such as sample handling, background nitrite contamination, and epithelial differentiation state. Therefore, appropriate controls and careful experimental design are required to ensure accurate interpretation of results.

<table><tbody><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole (DAPI)</td><td>ThermoFisher Scientific</td><td>62248</td><td>diluted 1:2000 for immunostaining</td></tr><tr><td>50 mL Centrifuge tube filter (0.22 &amp;micro;m)</td><td>CellTreat</td><td>229710</td><td>For preparation of sterile solutions</td></tr><tr><td>500 mL Filter units</td><td>ThermoFisher Scientific</td><td>566-0020</td><td>For sterile media preparation</td></tr><tr><td>Accutase</td><td>Sigma</td><td>A6964</td><td></td></tr><tr><td>Amphoterecin B</td><td>ThermoFisher Scientific</td><td>15290026</td><td></td></tr><tr><td>Bovine Pituitary Extract</td><td>ThermoFisher Scientific</td><td>13028014</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Cholera toxin</td><td>Sigma</td><td>C8052</td><td></td></tr><tr><td>Collagen I</td><td>ThermoFisher Scientific</td><td>A1048301</td><td></td></tr><tr><td>Confocal Microscope</td><td>Nikon</td><td></td><td>W1 Spinning disk Ti2 inverted microscope with Hamamatsu sCMOS camera</td></tr><tr><td>Corning Transwell inserts</td><td>Sigma</td><td>CLS3460</td><td></td></tr><tr><td>Diethylenetriamine NONOate (DETA-NONOate)</td><td>Cayman Chemical</td><td>82120</td><td></td></tr><tr><td>DMEM/F-12 media</td><td>ThermoFisher Scientific</td><td>11330032</td><td></td></tr><tr><td>DNAse I</td><td>Sigma</td><td>DN25</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F4135</td><td></td></tr><tr><td>Halt 1X Protease inhibitor cocktail</td><td>ThermoFisher Scientific</td><td>1860932</td><td></td></tr><tr><td>Ham&#039;s F1-12 Nutrient mix</td><td>ThermoFisher Scientific</td><td>11765054</td><td></td></tr><tr><td>Insulin solution</td><td>Sigma</td><td>I0516</td><td></td></tr><tr><td>Isoproterenol hydrochloride</td><td>Sigma</td><td>I6504</td><td></td></tr><tr><td>Keratinocyte Serum-free media</td><td>ThermoFisher Scientific</td><td>17005042</td><td></td></tr><tr><td>Murine Epidermal growth factor</td><td>Sigma</td><td>E4127</td><td></td></tr><tr><td>N(G)-Nitro-L-arginine methyl ester (L-NAME)</td><td>Sigma</td><td>N5751</td><td></td></tr><tr><td>NuSerum</td><td>Corning</td><td>355100</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Phosphate buffer saline (PBS)</td><td>ThermoFisher Scientific</td><td>10010023</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>ThermoFisher Scientific</td><td>23227</td><td></td></tr><tr><td>Pronase</td><td>Sigma</td><td>10165921001</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma</td><td>R2625</td><td></td></tr><tr><td>Rho kinase inhibitor (Y-27632 hydrochloride)</td><td>Cayman Chemical</td><td>TOM-C837Z37</td><td></td></tr><tr><td>RIPA buffer</td><td>Sigma</td><td>R0278</td><td></td></tr><tr><td>RPMI-1640 media</td><td>ThermoFisher Scientific</td><td>11875093</td><td></td></tr><tr><td>Sodium nitrite&amp;nbsp;</td><td>Sigma</td><td>S2252</td><td></td></tr><tr><td>Transferrin</td><td>Sigma</td><td>T8158</td><td></td></tr><tr><td>&amp;alpha;-Acetylated tubulin antibody (mouse)</td><td>Sigma</td><td>T6793</td><td>diluted 1:200 for immunostaining</td></tr><tr><td>&amp;alpha;-FoxJ1 antibody (mouse)</td><td>ThermoFisher Scientific</td><td>14-9965-80</td><td>diluted 1:200 for immunostaining</td></tr><tr><td>&amp;alpha;-mouseCy3 secondary antibody</td><td>ThermoFisher Scientific</td><td>A10521</td><td>diluted 1:1000 for immunostaining</td></tr><tr><td>&amp;gamma;-secretase inhibitor IX (DAPT)</td><td>Sigma</td><td>D5942</td><td></td></tr></tbody></table>

measurement of nitric oxide production in mouse tracheal epithelial cell cultures differentiated at the air liquid interface

Measurement of Nitric Oxide Production in Mouse Tracheal Epithelial Cell Cultures Differentiated at the Air Liquid Interface

Airway epithelial cultures differentiated at the air&#8211;liquid interface (ALI) provide a physiologically relevant model to study nitric oxide (NO) signaling in ciliated cells. Here, we describe a protocol to quantify NO production by measuring nitrite (NO&#8322;&#8315;), a stable oxidation product of NO, in samples collected from ALI-differentiated mouse tracheal epithelial cells. Apical washes, basal media and cell lysates are collected and analyzed using a triiodide-based chemiluminescence assay coupled to a nitric oxide analyzer (NOA). Upon injection into the reaction vessel, nitrite is chemically reduced to NO, which is detected by ozone-based chemiluminescence. Signal intensity is quantified and converted to picomoles of nitrite using a standard calibration curve, and values are normalized to total cellular protein content. Assay performance is validated using pharmacological modulation of NO levels. Upon treatment with the NO donor Diethylenetriamine NONOate (DETA-NONOate) and the NO synthase inhibitor N(&#969;)-Nitro-L-arginine methyl ester (L-NAME) we observe significant differences in nitrite levels across conditions. The most robust and reproducible signal is observed in apical washes, corresponding to the amount of NO released from the airway epithelium. This method reliably detects NO in samples from ALI-differentiated airway epithelia, providing an accurate in vitro platform to quantify NO production and model diseases associated with abnormal NO metabolism.

The overall goal of this procedure is to quantify nitric oxide (NO) levels in mouse tracheal epithelial cell (MTEC) cultures differentiated at air–liquid interface (ALI). Primary cells are isolated and differentiated according to established protocols27,28. Over the course of 21 days at ALI, the cells are differentiated into a pseudostratified airway epithelium containing ciliated cells27,28. Levels of ci...

This protocol describes a method to quantify nitric oxide (NO) production in airway epithelial cultures differentiated at the air&#8211;liquid interface (ALI) by measuring nitrite in apical secretions using a triiodide-based chemiluminescence assay.

Airway epithelial cultures differentiated at the air&#8211;liquid interface (ALI) provide a physiologically relevant model to study nitric oxide (NO) ...

measurement-nitric-oxide-production-mouse-tracheal-epithelial-cell

Research

JoVE Journal

Medicine

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium.  A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface  

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

In vitro Measurements of Tracheal Constriction Using Mice

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy.  Intravital microscopy is technically challenging in a moving organ such as the lung.   We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Isolation of Primary Mouse Lung Endothelial Cells

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

Here, we describe an in vitro model for isolating and differentiating murine airway epithelial cells, focusing on their acclimation to chronic cigarette smoke extract (CSE). The model could be utilized to comprehensively characterize the multi-omics impact of CSE, which possibly provides insights into the cellular responses under chronic smoke exposure.

In Vitro Model for Studying Differentiation and Changes of Multi-Omics on Murine Airway Epithelial Cells Stimulated with Cigarette Smoke Extract

Air-liquid interface culture is commonly used to develop pseudostratified airway epithelium by differentiating primary normal human bronchial epithelial cells that mimic the apical side of the lung airway. Here, we describe an easy protocol for determining its quality by monitoring its biophysical properties, such as ciliary function and membrane integrity.

Determining Ciliary Function and Membrane Impermeability of the Pseudostratified Lung Airway Epithelium

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions.  It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases.  Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

Analytical Techniques for Assaying Nitric Oxide Bioactivity

University of Maryland School of Medicine.  This protocol describes a method to quantify nitric oxide (NO) production in airway epithelial cultures differentiated at the air–liquid interface (ALI) by measuring nitrite in apical secretions using a triiodide-based chemiluminescence assay.

Measurement of Nitric Oxide Production in Mouse Tracheal Epithelial Cell Cultures Differentiated at the Air Liquid Interface

Explore More Videos