Cell-based assays using primary endothelial cells to study multiple steps in inflammation.
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 alpha (TNFalpha)-induced translocation of the transcription factor NF-kappaB from the cytoplasm into the nucleus 20 min after stimulation with TNFalpha. Two more assays monitor the expression of E-selectin and VCAM-1, 4 and 24 h after stimulation with TNFalpha. 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.
Mayer
Thomas
T
The Judith P. Sulzberger, MD, Columbia Genome Center, Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, USA.
Jagla
Bernd
B
Wyler
Michael R
MR
Kelly
Peter D
PD
Aulner
Nathalie
N
Beard
Matthew
M
Barger
Geoffrey
G
Többen
Udo
U
Smith
Deborah H
DH
Brandén
Lars
L
Rothman
James E
JE
D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D019040:E-Selectin / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology*; D004730:Endothelium, Vascular / Q000166:cytology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007249:Inflammation / Q000473:pathology*; D008856:Microscopy, Fluorescence / Q000379:methods*; D016328:NF-kappa B / Q000378:metabolism; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
D016207:Cytokines; D019040:E-Selectin; D016328:NF-kappa B; D034741:RNA, Small Interfering; D019010:Vascular Cell Adhesion Molecule-1