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

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.