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Plasmid DNA Preparation> | … 500,000 Caco-2 cells were suspended in 100 μL of buffer (Lonza #VCA-1002) supplemented with 264 pmol of the gRNA duplex, 10 μg of the Cas9 expression plasmid (GE Healthcare #U-005100-120), and 10 μg of the repair plasmid (Genscript, Note S1), followed by … | Get A Quote |
Nanotopographic materials provide special biophysical stimuli that can regulate epithelial tight junctions and their barrier function. Through the use of total internal reflection fluorescence microscopy of live cells, we demonstrated that contact of synthetic surfaces with defined nanotopography at the apical surface of epithelial monolayers increased paracellular permeability of macromolecules. To monitor changes in tight junction morphology in live cells, we fluorescently tagged the scaffold protein zonula occludens-1 (ZO-1) through CRISPR/Cas9-based gene editing to enable live cell tracking of ZO-1 expressed at physiologic levels. Contact between cells and nanostructured surfaces destabilized junction-assoc... More