title: Measuring intracellular calcium fluxes in high throughput mode.
abstract: The measurement of intracellular calcium fluxes in real time is widely applied within the pharmaceutical industry to measure the activation of G-protein coupled receptors (GPCRhyp;s), either for pharmacological characterisation or to screen for new surrogate ligands. Initially restricted to G(q) coupled GPCRs, the introduction of promiscuous and chimeric G-proteins has further widened the application of these assays. The development of new calcium sensitive dyes and assays has provided sensitive, homogeneous assays which can be readily applied to high throughput screening (HTS). In this paper we describe the full automation of this assay type using a fluorometric imaging plate reader (FLIPR ) integrated into a Beckman/Sagian system to establish a simple robotic system that is well suited for the current medium throughput screening in this area of lead discovery. Using a recently completed HTS we discuss important determinants for FLIPR  based screening, highlight some limitations of the current approach, and look at the requirements for future automated systems capable of keeping up with expanding compound files.
authors.affiliation: Pfizer Global Research and Development, Lead Discovery Technologies, IPC 580, Ramsgate Road, Sandwich CT13 9NJ, UK. andreas_sewing@sandwich.pfizer.com
authors.forename: Chris
authors.initials: C
authors.lastname: Chambers
authors.forename: Fiona
authors.initials: F
authors.lastname: Smith
authors.forename: Christine
authors.initials: C
authors.lastname: Williams
authors.forename: Sandra
authors.initials: S
authors.lastname: Marcos
authors.forename: Zhen Han
authors.initials: ZH
authors.lastname: Liu
authors.forename: Paul
authors.initials: P
authors.lastname: Hayter
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ciaramella
authors.forename: Wilma
authors.initials: W
authors.lastname: Keighley
authors.forename: Phil
authors.initials: P
authors.lastname: Gribbon
authors.forename: Andreas
authors.initials: A
authors.lastname: Sewing
mesh_terms: D001331:Automation; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005470:Fluorometry / Q000379:methods*; D019204:GTP-Binding Proteins / Q000819:agonists / Q000378:metabolism*; D006801:Humans; D011956:Receptors, Cell Surface / Q000819:agonists / Q000032:analysis* / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D011956:Receptors, Cell Surface; D019204:GTP-Binding Proteins; D002118:Calcium