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: Contribution to journalArticle

9 Citations (Scopus)

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)
Pages (from-to)266-283
Number of pages18
JournalMethods in Enzymology
Volume414
DOIs
StatePublished - 2006
Externally publishedYes

Fingerprint

Endothelial cells
Assays
Endothelial Cells
Inflammation
Tumor Necrosis Factor-alpha
Throughput
Preclinical Drug Evaluations
E-Selectin
Vascular Cell Adhesion Molecule-1
Human Umbilical Vein Endothelial Cells
Signal transduction
Eukaryotic Cells
Drug Discovery
Indirect Fluorescent Antibody Technique
Microscopy
Signal Transduction
Cytoplasm
Transcription Factors
Microscopic examination
Screening

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mayer, T., Jagla, B., Wyler, M. R., Kelly, P. D., Aulner, N., Beard, M., ... Rothman, J. E. (2006). Cell-Based Assays Using Primary Endothelial Cells to Study Multiple Steps in Inflammation. Methods in Enzymology, 414, 266-283. https://doi.org/10.1016/S0076-6879(06)14015-X

Cell-Based Assays Using Primary Endothelial Cells to Study Multiple Steps in Inflammation. / Mayer, Thomas; Jagla, Bernd; Wyler, Michael R.; Kelly, Peter D.; Aulner, Nathalie; Beard, Matthew; Barger, Geoffrey; Többen, Udo; Smith, Deborah H.; Brandén, Lars; Rothman, James E.

In: Methods in Enzymology, Vol. 414, 2006, p. 266-283.

Research output: Contribution to journalArticle

Mayer, T, Jagla, B, Wyler, MR, Kelly, PD, Aulner, N, Beard, M, Barger, G, Többen, U, Smith, DH, Brandén, L & Rothman, JE 2006, 'Cell-Based Assays Using Primary Endothelial Cells to Study Multiple Steps in Inflammation', Methods in Enzymology, vol. 414, pp. 266-283. https://doi.org/10.1016/S0076-6879(06)14015-X
Mayer, Thomas ; Jagla, Bernd ; Wyler, Michael R. ; Kelly, Peter D. ; Aulner, Nathalie ; Beard, Matthew ; Barger, Geoffrey ; Többen, Udo ; Smith, Deborah H. ; Brandén, Lars ; Rothman, James E. / Cell-Based Assays Using Primary Endothelial Cells to Study Multiple Steps in Inflammation. In: Methods in Enzymology. 2006 ; Vol. 414. pp. 266-283.
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