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.