Automated Plate Reader-Based Assays of Light-Activated GPCRs

Elliot J. Gerrard, Alexandra-Madelaine Tichy, Harald Janovjak

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

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 Ca2+ 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.

Original languageEnglish
Title of host publicationOptogenetics
Subtitle of host publicationMethods and Protocols
EditorsArmin Baumschlager
Place of PublicationNew York, NY
PublisherSpringer
Chapter16
Pages217-229
Number of pages13
ISBN (Electronic)978-1-0716-4047-0
ISBN (Print)978-1-0716-4046-3
DOIs
Publication statusPublished - 2025

Publication series

NameMethods in molecular biology (Clifton, N.J.)
PublisherHumana Press
Volume2840
ISSN (Print)1064-3745

Keywords

  • Automation
  • Cell signaling
  • Opsin
  • Optogenetics
  • OptoXR
  • Synthetic biology

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