For paracellular permeability, Caco-2 cells were plated on a transwell insert for 14 days. Lucifer Yellow (LY, Sigma) was added to the apical compartment. From the basolateral compartment, 50 μl sample was transferred in a 96-well plate, and the fluorescence was quantified using a fluorescence microtiter plate reader at 428/549 nm. For 3D permeability assay, colonic organoid or Caco-2 spheroid-like structures were incubated with 4 kDa fluorescein isothiocyanate-labeled dextran (FITC-4D, Sigma) for 24 h. The flux of FITC-4D from the basal to the luminal compartment (L/B ratio) was assessed using confocal microscopy19 (link). Confocal images were taken with Leica laser scanning confocal microscope (Leica Microsystems GmbH) and processed using ImageJ software61 (link).
Fazio A., Bordoni D., Kuiper J.W., Weber-Stiehl S., Stengel S.T., Arnold P., Ellinghaus D., Ito G., Tran F., Messner B., Henning A., Bernardes J.P., Häsler R., Luzius A., Imm S., Hinrichsen F., Franke A., Huber S., Nikolaus S., Aden K., Schreiber S., Sommer F., Natoli G., Mishra N, & Rosenstiel P. (2022). DNA methyltransferase 3A controls intestinal epithelial barrier function and regeneration in the colon. Nature Communications, 13, 6266.
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