Establishment of Intestinal Epithelial Cell Monolayers and Their Use in Calcium Switch Assay for Assessment of Intestinal Tight Junction Assembly.

Intestinal barrier function relies primarily on the assembly and integrity of tight junctions, which forms a size-selective barrier. This barrier restricts paracellular movement of solutes in various types of epithelia. Of note, extracellular Ca concentration affects tight junction assembly. Therefore, the removal and re-addition of Ca into cell culture medium of cultured intestinal epithelial cells causes destabilization and reassembly of tight junction to membrane periphery near apical surface, respectively. Based on this principle, the Ca-switch assay was established to investigate tight junction assembly in fully differentiated intestinal epithelial cells. This chapter provides a stepwise protocol for culture of intestinal epithelial cell monolayers using T84 cell line as an in vitro model and the Ca-switch assay for evaluating tight junction assembly.