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Abstract Mucus is an important component of airway host defenses that acts by enabling the trapping and clearance of infectious materials such as bacteria and viruses. It can be difficult, however, to design experiments that independently determine the extent to which mucus contributes to innate barrier functions in the lung. Here, we provide detailed protocols to collect mucus from human airway epithelial cultures and evaluate how the properties of mucus impact mucociliary transport and protection from viral infection. We include recommended test parameters depending on the specific research question as it relates to respiratory infectious diseases. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Analysis of mucociliary transport and ciliary beat frequency in HAE cultures Basic Protocol 2: Collection of mucus from HAE cultures Basic Protocol 3: Transplantation of mucus to HAE cultures and infection with virus 

Synthetic mucus barrier arrays are developed as a high-throughput screening platform for nanoparticle drug delivery applications. 

A method to extract gel-forming mucins from pig trachea is developed. These airway mucins form viscous gels resembling human airway mucus, making them a useful model for lung disease research. 

The release and accumulation of neutrophil extracellular traps (NETs) in the airway mucus barrier is prominent in cystic fibrosis. New biomaterial-based models are developed that mimic important aspects of NET-mediated pathobiology in cystic fibrosis.