Format

Send to

Choose Destination
Anal Biochem. 1996 Oct 15;241(2):254-9.

Development of a high volume screen to identify inhibitors of endothelial cell activation.

Author information

1
Sphinx Pharmaceuticals, Division of Eli Lilly and Company, Durham, North Carolina 27707, USA.

Abstract

We have developed a high throughput screen to identify inhibitors of endothelial cell activation using E-selection cell-surface expression as a marker. Endothelial cell activation is an important component of both acute and chronic inflammatory disease. Inhibitors of this process represent potential therapeutic agents. A cell culture system for primary human umbilical vein endothelial cells was generated, including an analysis of donor variability, passage number, seeding density, and cost. The effects of IL-1 beta, TNF alpha, LPS, and an LPS-conditioned plasma product on E-selectin expression were characterized. E-selectin expression on the surface of IL-1-stimulated endothelial cells was quantified with a direct ELISA on fixed cell monolayers. Automation of the ELISA necessitated identification of methods for cell fixation, liquid handling, and compound addition which would maintain the integrity of the cell monolayer. The ELISA is inexpensive, reproducible, and suitable for high throughput primary cell assays, supporting a screening rate of 10,000 compounds/ week. The method is compatible with a broad chemical diversity, and the cellular format provides early information on the cellular uptake and cytotoxicity of compounds. We describe a screening paradigm which allowed us to identify inhibitors of endothelial cell activation and simultaneously discriminate their activity from their cytotoxic effects.

PMID:
8921195
DOI:
10.1006/abio.1996.0407
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center