Autori: Edwards BS, Young SM, Oprea TI, Bologa CG, Prossnitz ER, Sklar LA

Editorial: Nature Publishing Group, Nature Protocols, 1(1), p.59 - 66, 2006.

Rezumat:

The formylpeptide receptor (FPR) family of G protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here we describe a high-throughput flow cytometry screening approach that has successfully identified multiple families of previously unknown FPR ligands. The assay detects active structures that block the binding of a fluorescent ligand to membrane FPR of intact cells, thus detecting both agonists and antagonists. It is homogeneous in that assay reagents are added in sequence and the wells are subsequently analyzed without intervening wash steps. Microplate wells are routinely processed at a rate of 40 wells per minute, requiring a volume of only 2 mul to be sampled from each. This screening approach has recently been extended to identify a high-affinity, selective agonist for the intracellular estrogen-binding G protein&-coupled receptor GPR30. With the development of appropriate assay reagents, it may be generally adaptable to a wide range of receptors. The total time required for the assay ranges between 1.5 and 2.5 h. The time required for flow cytometry analysis of a 96-well plate at the end of the procedure is less than 2.5 min. By comparison, manual processing of 96 samples will typically require 40-50 min, and a fast commercial automated sampler processes 96-well plates in less than 15 min, requiring the aspiration of 22 mul per sample for an analysis volume of 2 mul.

Cuvinte cheie: Formyl Peptide Receptor, GPCR, High-Throughput Screening

URL: http://www.nature.com/nprot/journal/v1/n1/abs/nprot.2006.9.html