High-Throughput Assays to Measure Intracellular Ca2+ Mobilization in Cells that Express Recombinant S1P Receptor Subtypes
Intracellular Ca2+ 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 Ca2+ indicator dye Fura-2-AM. Changes in intracellular Ca2+ levels in response to S1P or test compounds are detected using a FlexStation II scanning fluorometer with integrated fluidics transfer capabilities.
Key wordsCalcium assay G-protein coupled receptor Sphingosine-1-phosphate Lysophospholipid FlexStation EDG receptor
We thank Dr. Bruce Conklin (University of California, San Francisco) for generously providing chimeric G-protein expression plasmids and Dr. Edward Goetzl (University of California, San Francisco) for the stable S1P receptor cell lines. This work was supported by NIH grant CA-092160.
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