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T-Cell Trafficking pp 155–168Cite as

Utilizing Lentiviral Gene Transfer in Primary Endothelial Cells to Assess Lymphocyte-Endothelial Interactions

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1591))

Abstract

A major impediment when studying primary human endothelial cell function is the resistance of these cells to gene transfer. Here we describe methods for transferring genes into primary endothelial cells prior to incorporation into a static adhesion assay to analyze the adhesion and migration of isolated lymphocytes. Human embryonic kidney (HEK)-293T (HEK-293 cells expressing the large T-antigen of simian virus 40) cells are initially transfected with plasmids containing the lentiviral packaging and envelope genes and the target sequence, such as a gene of interest or short hairpin loop RNA (shRNA). These cells produce lentivirus packaged with this target sequence and are used to transduce primary human umbilical vein endothelial cells (HUVECs). Human peripheral blood lymphocytes (PBLs) isolated from venous blood are co-incubated with lentivirally transduced cytokine-stimulated endothelial cells to assess lymphocyte adhesion in a static adhesion assay. Direct observations of lymphocyte adhesion and migration over a time course can also be made. In general, lentiviral transduction of primary endothelial cells provides an invaluable system to manipulate gene expression levels when studying the cellular adhesion dynamics that regulate leukocyte adhesion and extravasation.

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Author information

Authors and Affiliations

  1. School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham, B15 2TT, UK

    Jasmeet S. Reyat B.Sc., Michael G. Tomlinson D.Phil. & Peter J. Noy Ph.D.

Authors
  1. Jasmeet S. Reyat B.Sc.
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  2. Michael G. Tomlinson D.Phil.
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  3. Peter J. Noy Ph.D.
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Corresponding author

Correspondence to Peter J. Noy Ph.D. .

Editor information

Editors and Affiliations

  1. Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom

    George Edward Rainger

  2. Institute of Inflammation and Ageing, College of Medicine and Dental Sciences, University of Birmingham, Birmingham, United Kingdom

    Helen M. Mcgettrick

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Reyat, J.S., Tomlinson, M.G., Noy, P.J. (2017). Utilizing Lentiviral Gene Transfer in Primary Endothelial Cells to Assess Lymphocyte-Endothelial Interactions. In: Rainger, G., Mcgettrick, H. (eds) T-Cell Trafficking. Methods in Molecular Biology, vol 1591. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6931-9_11

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  • DOI: https://doi.org/10.1007/978-1-4939-6931-9_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6929-6

  • Online ISBN: 978-1-4939-6931-9

  • eBook Packages: Springer Protocols

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