Open AccessTranscytosis Assay for Transport of Glycosphingolipids across MDCK-II Cells
Author(s) -
María García-Castillo,
Wayne I. Lencer,
Daniel J.F. Chinnapen
Publication year - 2018
Publication title -
bio-protocol
Language(s) - English
Resource type - Journals
ISSN - 2331-8325
DOI - 10.21769/bioprotoc.3049
Subject(s) - transcytosis , microbiology and biotechnology , glycosphingolipid , endocytic cycle , secretion , peptide , endocytosis , cell , vesicular transport protein , in vitro , biology , chemistry , biochemistry , vesicle , membrane
Absorption and secretion of peptide and protein cargoes across single-cell thick mucosal and endothelial barriers occurs by active endocytic and vesicular trafficking that connects one side of the epithelial or endothelial cell (the lumen) with the other (the serosa or blood). Assays that assess this pathway must robustly control for non-specific and passive solute flux through weak or damaged intercellular junctions that seal the epithelial or endothelial cells together. Here we describe an in vitro cell culture Transwell assay for transcytosis of therapeutic peptides linked covalently to various species of the glycosphingolipid GM1. We recently used this assay to develop technology that harnesses endogenous mechanism of lipid sorting across epithelial cell barriers to enable oral delivery of peptide and protein therapeutics.