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

Summary

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.

Abstract

Airway epithelial cultures differentiated at the air–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 (NO₂⁻), 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(ω)-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.

Introduction

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 asthma^1,2, while drastically reduced in CF and PCD^3,4. These diseases commonly exhibit abnormalities in airway epithelial motile cilia, the key effectors of mucociliary clearance⁵. Because NO and its downstream signali....

Protocol

All animal procedures were reviewed and approved by the University of Maryland Institutional Animal Care and Use Committee (Protocol # AUP–00004632).

NOTE: The protocol requires approximately 2 days for isolation of mice tracheal epithelial cells (MTEC), followed by 7–10 days for expansion, 5–6 days for proliferation in transwell inserts, 21 days for differentiation (time needed to acquire sufficient multi–ciliated cells), 24 h of drug treatment and one additional day for sample collection, NOA calibration, injections and data acquisition.

1. Preparation of media and solutions

Results

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 protocols^27,28. Over the course of 21 days at ALI, the cells are differentiated into a pseudostratified airway epithelium containing ciliated cells^27,28. Levels of ci.......

Discussion

Multi–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 homeostasis^6,7,8. NO is a highly reactive molecule and even small changes in NO levels are associated with airway diseases^1,2,3,

Materials

List of materials used in this article

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