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Heparin‐avidin interaction interferes with the XPerT permeability assay
Author(s) -
Furman William,
Garrett Joshua
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.709.8
Subject(s) - biotinylation , avidin , heparin , permeability (electromagnetism) , chemistry , gelatin , endothelial stem cell , endothelium , biophysics , in vitro , monolayer , cell culture , microbiology and biotechnology , biochemistry , medicine , biology , membrane , genetics
The endothelium is a monolayer of cells that line the vasculature and act as a semi‐permeable barrier controlling what passes from the lumen of blood vessels into the interstitium. Acute changes in endothelial permeability are commonly seen during inflammation. There are several in vitro assays which allow the researcher to look at changes in endothelial permeability under various inflammatory conditions, one of which is known as the XPerT assay. In this assay, endothelial permeability is assessed using fluorescence microscopy and is based on high affinity interactions between ligand (Alexa fluor‐488‐conjugated avidin) added in the culture medium, which permeates cell monolayer to reach substrate‐bound acceptor (biotinylated gelatin). Interestingly, heparin is known to strongly bind avidin and is also commonly found in different formulations of endothelial media in wildly differing concentrations. From this, we hypothesized that media with high concentration of heparin would interfere with the XPerT assay. To test this, we added increasing amounts of heparin to the XPerT assay and found that high doses impeded the ability of the avidin to interact with the biotinylated gelatin. Furthermore, we found that when high doses of heparin were present, the avidin was more likely to bind directly to the endothelial cells and not the biotinylated gelatin. Taken together, our data suggests that using a low heparin containing endothelial medium is optimal for using the XPerT assay to test endothelial permeability. Support or Funding Information This work was supported in part through the Hartwick College Faculty Research Grants Program. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .