Cell-Based Assays Using Primary Endothelial Cells to Study Multiple Steps in Inflammation

Thomas Mayer, Bernd Jagla, Michael R. Wyler, Peter D. Kelly, Nathalie Aulner, Matthew Beard, Geoffrey Barger, Udo Többen, Deborah H. Smith, Lars Brandén, James E. Rothman

Research output: Chapter in Book/Report/Conference proceedingChapter

9 Scopus citations

Abstract

Cell-based assays are powerful tools for drug discovery and provide insight into complex signal transduction pathways in higher eukaryotic cells. Information gleaned from assays that monitor a cellular phenotype can be used to elucidate the details of a single pathway and to establish patterns of cross talk between pathways. By selecting the appropriate cell model, cell-based assays can be used to understand the function of a specific cell type in a complex disease process such as inflammation. We have used human umbilical vein endothelial cells to establish three cell-based, phenotypic assays that query different stages of a major signaling pathway activated in inflammation. One assay analyzes the tumor necrosis factor α (TNFα)-induced translocation of the transcription factor NF-κB from the cytoplasm into the nucleus 20 min after stimulation with TNFα. Two more assays monitor the expression of E-selectin and VCAM-1, 4 and 24 h after stimulation with TNFα. Indirect immunofluorescence and high-throughput automated microscopy were used to analyze cells. Imaging was performed with the IN Cell Analyzer 3000. All assays proved to be highly robust. Z′ values between 0.7 and 0.8 make each of the three assays well suited for use in high-throughput screening for drug or probe discovery.

Original languageEnglish (US)
Title of host publicationMeasuring Biological Responses with Automated Microscopy
PublisherAcademic Press Inc.
Pages266-283
Number of pages18
ISBN (Print)0121828190, 9780121828196
DOIs
StatePublished - Jan 1 2006

Publication series

NameMethods in Enzymology
Volume414
ISSN (Print)0076-6879

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Mayer, T., Jagla, B., Wyler, M. R., Kelly, P. D., Aulner, N., Beard, M., Barger, G., Többen, U., Smith, D. H., Brandén, L., & Rothman, J. E. (2006). Cell-Based Assays Using Primary Endothelial Cells to Study Multiple Steps in Inflammation. In Measuring Biological Responses with Automated Microscopy (pp. 266-283). (Methods in Enzymology; Vol. 414). Academic Press Inc.. https://doi.org/10.1016/S0076-6879(06)14015-X