Automated Imaging Assay for Characterizing Ca2+ Flux with R-GECO Biosensor

GPCR activation was kinetically monitored using a Ca²⁺ biosensor that was transfected along with the human muscarinic M1 receptor into HEK 293 cells. Rapid Ca²⁺ flux was evident upon carbachol stimulation that peaked 1 second after injection, followed by a decay back to baseline fluorescence over 90 seconds. Imaging also allowed for the monitoring of single cells within the cell population in the field of view where differences in kinetics can be assessed. Suitable assay performance was achieved using a % Responders readout (% of cells responding to carbachol stimulation) that provided z’ values above 0.5 and consistent pharmacology.