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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. 

Abstract Prior work suggests influenza A virus (IAV) crosses the airway mucus barrier in a sialic acid‐dependent manner through the actions of the viral envelope proteins, hemagglutinin, and neuraminidase. However, host and viral factors that influence how efficiently mucus traps IAV remain poorly defined. In this work, how the physicochemical properties of mucus influence its ability to effectively capture IAV is assessed using fluorescence video microscopy and multiple particle tracking. Our studies suggest an airway mucus gel layer must be produced with virus‐sized pores to physically constrain IAV. While sialic acid binding by IAV may improve mucus trapping efficiency, sialic acid binding preference is found to have little impact on IAV mobility and the fraction of viral particles expected to penetrate the mucus barrier. Further, synthetic polymeric hydrogels engineered with mucus‐like architecture are similarly protective against IAV infection despite their lack of sialic acid decoy receptors. Together, this work provides new insights on mucus barrier function toward IAV with important implications on innate host defense and transmission of respiratory viruses.