Automated Plate Reader-Based Assays of Light-Activated GPCRs.

In the emerging field of optogenetics, light-sensitive G-protein coupled receptors (GPCRs) allow for the temporally precise control of canonical cell signaling pathways. Expressing, stimulating, and measuring the activity of light-sensitive GPCRs (e.g., opsins or chimeric OptoXRs) in mammalian cells is a nontrivial task as many standard assay practices are not compatible with light-sensitive molecular tools. In this chapter, we present a method for quantifying opsin activity in automated plate reader-based assays without the need for additional optical hardware (i.e., light sources). The protocol is applied to assess cAMP levels downstream of a chimeric OptoXR but can be expanded to other opsins and second messengers, such as Ca mobilization. We describe how the internal optical components in commonly available plate readers can be utilized to both activate and detect kinetic and dose-response relationships, as well as provide general guidance for optimizing assays with light-sensitive molecular tools.