Epithelial tight junctions create a boundary between the apical and basolateral cell surface domains, thereby regulating diffusion along the paracellular pathway. They also form a semi-permeable paracellular gate that restricts diffusion in a charge- and size-selective manner. Since the molecular mechanisms of size-selective diffusion are poorly understood, studying barrier formation by endothelia and epithelia is important, especially in treating chronic inflammations or cancer.
Paracellular permeability of hydrophilic tracers can be monitored with compounds that are labelled fluorescently, such as dextrans. Herein, fluorescently labelled dextrans (4kD FITC dextran and 70 kD Rhodamine dextrans) are used as tracers since different sizes can be analysed by the same detection method; moreover, the use of both FITC and Rhodamine labelled dextrans allows for the analysis of two different tracers in the same culture.
Using the BMG LABTECH microplate reader for these experiments allows quick and consistent fluorescence measurements in either kinetic or endpoint format.