Quantifying the unfolded protein response using a nontoxic genetically-encoded fluorescent sensor, live-cell imaging, and label-free cellular impedance.

• Genetically-encoded, single-color fluorescent biosensor detects β-arrestin signaling in living cells.
• β-arrestin sensor multiplexed with Gq sensor simultaneously detects arrestin and G-protein signaling.
• Kinetic analysis of arrestin and G-protein signaling on the CLARIOstar® reliably detects agonist bias.

• Genetically-encoded biosensors from Montana Molecular allow for detection of GPCR signaling.
• Robust Z′ measurements, kinetic readouts, and dose response

Fluorescent dyes and biosensors can be used to monitor changes in second messenger levels, including Ca²⁺ and cAMP, in response to GPCR activation. Here we describe a live-cell imaging based approach to detect GPCR activation using the Lionheart FX automated imager and Gen5 image analysis software. This method provides a large assay window and improved sensitivity over methods relying on total fluorescence intensity measurements.