Automated Plate Reader-Based Assays of Light-Activated GPCRs

Area of Science:

• Optogenetics
• Molecular Biology
• Cell Signaling

Background:

• Light-sensitive G-protein coupled receptors (GPCRs) offer precise temporal control over cellular signaling pathways.
• Standard assay practices are often incompatible with light-sensitive molecular tools like opsins and chimeric OptoXRs.
• Quantifying the activity of these tools in mammalian cells presents a significant challenge.

Purpose of the Study:

• To develop and present a method for quantifying opsin activity in automated plate reader-based assays.
• To demonstrate the utility of internal plate reader optics for activating and detecting light-sensitive molecular tools.
• To provide guidance for optimizing assays involving light-sensitive molecular tools.

Main Methods:

• Utilizing internal optical components of common plate readers for both activation and detection.
• Applying the protocol to assess cyclic adenosine monophosphate (cAMP) levels downstream of a chimeric OptoXR.
• Adapting the method for other opsins and second messengers like calcium (Ca2+) mobilization.

Main Results:

• Successful quantification of opsin activity without external optical hardware.
• Demonstration of kinetic and dose-response relationship detection using standard plate readers.
• Validation of the protocol for cAMP level assessment.

Conclusions:

• A practical method is established for assaying light-sensitive GPCRs in mammalian cells using readily available plate readers.
• This approach simplifies optogenetic experiments by eliminating the need for specialized light sources.
• The protocol is versatile and can be extended to various opsins and signaling pathways.