Function Therapeutics; Department of Chemistry, Marquette University; School of Medicine, Washington University.
Function Therapeutics; Department of Chemistry, Marquette University;
J Vis Exp. 2024 May 24(207). doi: 10.3791/66507.
Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of Gαq signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.
通过使用细胞内荧光钙结合染料和成像仪器，可以快速高通量地监测细胞内钙离子浓度的变化，这些仪器可以同时测量多达 1536 个孔的荧光发射。然而，与广泛可用的逐个扫描孔的平板读数仪相比，这些仪器要昂贵得多，并且维护起来也更具挑战性。本文描述了一种优化的平板读数仪测定法，用于测量内皮细胞系（EA.hy926）中蛋白酶激活受体（PAR）驱动的 Gαq 信号激活以及随后使用钙结合染料 Fluo-4 进行的钙动员。该测定法已用于表征一系列 PAR 配体，包括在 Dockendorff 实验室中发现的靶向 PAR1 的变构抗炎“parmodulin”配体。该方案不需要自动化液体处理机，并允许在 96 孔板中进行中等通量的 PAR 配体筛选，并且应该适用于研究其他引发钙动员的受体。
