Glyco-TRAPP: A real-time glycocalyx permeability assay for assessing transmembrane mucin barrier function in live and fixed tissues.

A glycocalyx permeability assay for in situ assessment of transmembrane mucin barrier function ABSTRACT: The small intestine lacks a dense, attached mucus layer and instead relies on an apical cell surface glycocalyx as an alternative barrier mechanism to regulate host-microbiota interactions. However, quantitatively assessing the integrity of the glycocalyx barrier in a physiologically relevant setting has remained challenging. Here, we introduce the Glyco-TRAPP assay, a live imaging-based approach that enables direct, quantitative measurement of glycocalyx permeability and bacteria-epithelium interactions in intact intestinal tissue. By enabling direct functional assessment of barrier integrity in both human biopsies and experimental models, Glyco-TRAPP provides a powerful tool to uncover how molecular defects at the cell surface, compromising for example transmembrane mucin expression, localization, and glycosylation, impact glycocalyx barrier function. Glyco-TRAPP provides a functional readout that complements conventional structural or molecular analyses.