Measuring intracellular calcium fluxes in high throughput mode.
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.
Chambers
Chris
C
Pfizer Global Research and Development, Lead Discovery Technologies, IPC 580, Ramsgate Road, Sandwich CT13 9NJ, UK. andreas_sewing@sandwich.pfizer.com
Smith
Fiona
F
Williams
Christine
C
Marcos
Sandra
S
Liu
Zhen Han
ZH
Hayter
Paul
P
Ciaramella
Giuseppe
G
Keighley
Wilma
W
Gribbon
Phil
P
Sewing
Andreas
A
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
D011956:Receptors, Cell Surface; D019204:GTP-Binding Proteins; D002118:Calcium