Gerrard Elliot J, Tichy Alexandra-Madelaine, Janovjak Harald
Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
Australian Regenerative Medicine Institute (ARMI), Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia.
Methods Mol Biol. 2025;2840:217-229. doi: 10.1007/978-1-0716-4047-0_16.
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.
在新兴的光遗传学领域，光敏感的G蛋白偶联受体（GPCRs）能够对经典细胞信号通路进行时间精确控制。在哺乳动物细胞中表达、刺激和测量光敏感GPCRs（如视蛋白或嵌合OptoXRs）的活性并非易事，因为许多标准检测方法与光敏感分子工具不兼容。在本章中，我们介绍了一种在基于自动酶标仪的检测中量化视蛋白活性的方法，无需额外的光学硬件（即光源）。该方案用于评估嵌合OptoXR下游的cAMP水平，但可扩展到其他视蛋白和第二信使，如钙离子动员。我们描述了如何利用常用酶标仪中的内部光学组件来激活和检测动力学及剂量反应关系，以及为使用光敏感分子工具优化检测提供一般指导。
