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Abstract
Immunoassays are valuable and widely used tools in the biological sciences. In drug discovery research, immunoassays are used to measure secreted cytokines in the tissue culture supernatants of treated cells. However, scientists often avoid immunoassays in high throughput screening (HTS) because they require cumbersome manipulations. Applied Biosystems has developed a singlestep, mix-and-read bead-based immunoassay, termed FLISA (fluorescence- linked immunosorbent assay), for the FMAT 8100 HTS System. The instrument is a macro-confocal scanner that enables mix-and-read, bead- and live cell-based HTS assays in 96- and 384-well formats. Furthermore, multiplexed assays, based on color or bead size, can be performed in a single well.
The unique features of the FMAT system allow bead-based FLISAs to be performed in the same well in which tissue culture cells are grown. A simple multiplexed protocol can simultaneously quantitate secreted cytokines and detect the upregulation of a cell surface marker on human umbilical vein endothelial cells (HUVEC) in a mix-and-read assay. After induction with IL-1 or TNF-, the cells are either stained with CentriRed Stain, a nucleic acid staining dye from Applied Biosystems, or with an anti-ICAM-1 antibody to measure upregulation of the cell surface adhesion molecule. At the same time, reagents used in the FLISA are added for quantification of secreted IL-8. Using the FMAT system for simultaneous measurement of secreted metabolites and cell surface markers simplifies data collection and enhances information obtained from a high throughput screen.
Introduction
Cytokines are important mediators in the inflammatory response of vascular endothelial cells (EC). Their presence promotes the secretion of additional pro-inflammatory cytokines and expression of cell-surface adhesion molecules. Upon activation with I
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