title: High-throughput assays to measure intracellular Ca²⁺ mobilization in cells that express recombinant S1P receptor subtypes.
abstract: Intracellular Ca(2+) mobilization is a useful readout to screen for agonists or antagonists of G-protein -coupled receptors (GPCRs). Here, we describe methods to conduct high-throughput screening of stably or transiently transfected HTC4 cells expressing the individual S1P1-5 receptor subtypes. The cells are grown in 96-well plates and loaded with the cell permeable fluorescent Ca(2+) indicator dye Fura-2-AM. Changes in intracellular Ca(2+) levels in response to S1P or test compounds are detected using a FlexStation II scanning fluorometer with integrated fluidics transfer capabilities.
authors.affiliation: Department of Physiology, The University of Tennessee Health Science Center, Memphis, TN, USA. wvalentine@uthsc.edu
authors.forename: William J
authors.initials: WJ
authors.lastname: Valentine
authors.forename: Gabor
authors.initials: G
authors.lastname: Tigyi
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D020013:Calcium Signaling; D005470:Fluorometry; D016257:Fura-2 / Q000031:analogs & derivatives; D057166:High-Throughput Screening Assays; D051381:Rats; D049349:Receptors, Lysosphingolipid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D049349:Receptors, Lysosphingolipid; D011994:Recombinant Proteins; C049925:fura-2-am; D002118:Calcium; D016257:Fura-2