{"title":"Protocol for detecting endogenous GPCR activity in primary cell cultures using ONE-GO biosensors.","authors":"Remi Janicot, Mikel Garcia-Marcos","doi":"10.1016/j.xpro.2024.103355","DOIUrl":null,"url":null,"abstract":"<p><p>ONE vector G protein Optical (ONE-GO) biosensors can measure the activity of endogenously expressed G protein-coupled receptors (GPCRs) in primary cells. By detecting G proteins that belong to all four families (G<sub>s</sub>, G<sub>i/o</sub>, G<sub>q/11</sub>, G<sub>12/13</sub>) across cell types, these biosensors provide high experimental versatility. We first describe steps to express ONE-GO biosensors in primary cells using lentiviral transduction. We then detail how to carry out measurements and subsequent analysis to quantify changes in bioluminescence resonance energy transfer (BRET) reporting on endogenous GPCR activity. For complete details on the use and execution of this protocol, please refer to Janicot et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103355"},"PeriodicalIF":1.3000,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480228/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"STAR Protocols","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.xpro.2024.103355","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/10/1 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
ONE vector G protein Optical (ONE-GO) biosensors can measure the activity of endogenously expressed G protein-coupled receptors (GPCRs) in primary cells. By detecting G proteins that belong to all four families (Gs, Gi/o, Gq/11, G12/13) across cell types, these biosensors provide high experimental versatility. We first describe steps to express ONE-GO biosensors in primary cells using lentiviral transduction. We then detail how to carry out measurements and subsequent analysis to quantify changes in bioluminescence resonance energy transfer (BRET) reporting on endogenous GPCR activity. For complete details on the use and execution of this protocol, please refer to Janicot et al.1.
ONE 向量 G 蛋白光学（ONE-GO）生物传感器可以测量原代细胞中内源性表达的 G 蛋白偶联受体（GPCR）的活性。通过检测细胞类型中所有四个家族（Gs、Gi/o、Gq/11、G12/13）的 G 蛋白，这些生物传感器提供了高度的实验通用性。我们首先介绍了利用慢病毒转导在原代细胞中表达 ONE-GO 生物传感器的步骤。然后，我们详细介绍了如何进行测量和后续分析，以量化生物发光共振能量转移 (BRET) 报告内源性 GPCR 活性的变化。有关本方案使用和执行的完整细节，请参阅 Janicot 等人的文章1。
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