Background

The endothelial barrier is a well-regulated structure, which maintains a minimal and selective permeability to fluid and molecules under normal physiological conditions (Komarova and Malik, 2010). The disruption of the endothelial barrier occurs during exposure to inflammatory cytokines, pathogen infection, or cancer metastasis, which induces the disruption of cytoskeleton, cell-cell junction, or cell-to-matrix attachments. The increase in vascular permeability is an important feature in many diseases, including ischemia-reperfusion injury, sepsis, viral hemorrhagic fevers and cancers. To screen which molecules modulate vascular permeability, it is necessary to establish in vitro systems to test endothelial permeability before expanding to animal studies. There are two available systems to test endothelial permeability in vitro, transwell permeability assay and electrical impedance sensing devices (Bischoff et al., 2016). The transwell permeability assays directly detect the penetration of macromolecules and the electrical impedance sensing devices measure the cell layer’s tightness for ion flow. Basically, molecules which can be detected by a spectrometer-based absorbance reader can be used in the transwell permeability assay. As a result, the materials required for this assay are relatively easy to prepare. For the electrical impedance sensing assay, we used the xCELLigence Real-Time Cell Analysis (RTCA) systems to measuring endothelial permeability in a 96-well microplate. Compared to transwell permeability assay, electrical impedance sensing device is more sensitive, and is suitable for time-lapse tracking. However, it is also more expensive, and it may not accurately reflect the penetration of molecules through cell-cell junction. As a result, it is more accurate to apply both systems in parallel. Here, we show the protocol for using these two methods to measure the dengue virus nonstructural protein 1-induced endothelial hyperpermeability in vitro (Chen et al., 2016).