使用定量高通量测定法测量 G 蛋白偶联受体诱导的细胞内钙信号。

Measuring GPCR-Induced Intracellular Calcium Signaling Using a Quantitative High-Throughput Assay.

机构信息

Faculty of Medical and Health Sciences, Department of Molecular Medicine and Pathology, School of Medical Sciences, The University of Auckland, Auckland, New Zealand.

出版信息

Methods Mol Biol. 2025;2861:3-22. doi: 10.1007/978-1-0716-4164-4_1.

DOI:10.1007/978-1-0716-4164-4_1

PMID:39395093

Abstract

Alterations in intracellular calcium are integral to signal transduction pathways for many G-protein-coupled receptors, but this signaling is not well studied. This is mostly due to a lack of reliable, robust, high-throughput, quantitative methods to monitor intracellular calcium concentrations in live cells. Recently, we developed a reliable, robust, quantitative method to measure intracellular calcium levels in which HEK293 cell suspensions loaded with Fura-2/AM are placed in 96-well plates. Minimum and maximum intracellular calcium levels, which are required for converting fluorescence into calcium concentrations, are calibrated using EGTA to chelate calcium and ionomycin to load calcium into cells, respectively. Fluorescence is monitored with a PHERAstar FS plate reader. We provide a detailed method for this high-throughput assay that can be used to quantitate intracellular calcium in endogenous and exogenously (stable or transient) expressed GPCRs in HEK293 cells.

摘要

细胞内钙离子的改变是许多 G 蛋白偶联受体信号转导途径的重要组成部分，但这种信号转导尚未得到很好的研究。这主要是因为缺乏可靠、稳健、高通量、定量的方法来监测活细胞内的细胞内钙离子浓度。最近，我们开发了一种可靠、稳健、定量的方法来测量细胞内钙水平，其中将加载 Fura-2/AM 的 HEK293 细胞悬浮液置于 96 孔板中。使用 EGTA 螯合钙和离子霉素将钙加载到细胞中，分别校准最小和最大细胞内钙水平，这是将荧光转化为钙浓度所必需的。荧光通过 PHERAstar FS 平板读数仪进行监测。我们提供了一种详细的高通量测定方法，可用于定量 HEK293 细胞中内源性和外源性（稳定或瞬时）表达的 GPCR 中的细胞内钙。