Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

Introduce plasmid DNA encoding the target orphan GPCR into HEK293T cells using standard transfection reagents. Allow 24–48 hours for transgene expression before assay.

Load transfected cells with calcium-sensitive fluorescent dye, expose to test ligands at varying concentrations, and measure intracellular calcium flux via fluorescence intensity changes to identify receptor activation.

Use high-throughput plate reader or automated screening device to dispense ligand solutions, acquire real-time fluorescence data across multiple wells simultaneously, and generate concentration-response curves.

Plot dose-response data, calculate EC₅₀ values, and confirm functional ligand identification by demonstrating saturable, concentration-dependent receptor activation consistent with pharmacological principles.