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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.

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

  • 实验方案

  • 发表日期：

    2025年

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  • Shree S. Kumar,
  • Kathleen G. Mountjoy

摘要

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.