TY - JOUR A1 - Bartosova, Maria A1 - Ridinger, David A1 - Marinovic, Iva A1 - Heigwer, Jana A1 - Zhang, Conghui A1 - Levai, Eszter A1 - Westhoff, Jens H. A1 - Schaefer, Franz A1 - Terjung, Stefan A1 - Hildenbrand, Georg A1 - Krunic, Damir A1 - Bestvater, Felix A1 - Hausmann, Michael A1 - Schmitt, Claus Peter A1 - Zarogiannis, Sotirios G. T1 - An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept JF - International Journal of Molecular Sciences N2 - Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell–cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia. KW - Endothelzelle KW - Nabelvene Y1 - 2021 U6 - https://doi.org/https://doi.org/10.3390/ijms22158178 SN - 1422-0067 VL - 22 IS - 15 PB - MDPI AG ER -