Protocol for characterizing airway epithelial ciliary beating and mucociliary transport using image processing and particle imaging velocimetry

Lung epithelial cilia play a crucial role in respiratory health by driving mucociliary transport (MCT) and clearing inhaled pathogens and materials. Here, we present a protocol for measuring both ciliary beat frequency (CBF) and MCT in mucociliary human small airway epithelial cells. We describe steps for configuring experimental setups, collecting CBF and MCT data, analyzing datasets, and performing post-acquisition analysis. This protocol is adaptable to mucociliary cells from other lung regions and to mouse tracheal epithelial cells. For complete details on the use and execution of this protocol, please refer to Thomas et al.¹.