Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader

Document Type

Article

Publication Date

5-2024

Publisher

JoVE

Source Publication

Journal of Visualized Experiments

Source ISSN

1940-087X

Original Item ID

DOI: 10.3791/66507

Abstract

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.

Comments

Journal of Visualized Experiments, Vol. 207 (May 2024). DOI.

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