Анализ флуоресценции кальция с двойным добавлением для высокопроизводительного скрининга рекомбинантных рецепторов, связанных с G-белком

Overview

This study presents a high-throughput, intracellular calcium fluorescence assay designed for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs). The assay enables the identification of both agonists and antagonists using a dual-addition method with CHO-K1 cells expressing the kinin receptor from Rhipicephalus microplus.

Key Study Components

Area of Science

• Neuroscience
• Pharmacology
• Cell Biology

Background

• G protein-coupled receptors (GPCRs) play a crucial role in cellular signaling.
• Calcium signaling is a key pathway for many GPCRs.
• High-throughput screening (HTS) methods are essential for drug discovery.
• The dual addition assay allows simultaneous detection of agonists and antagonists.

Purpose of Study

• To develop a robust assay for screening small molecule ligands of GPCRs.
• To optimize conditions for reproducibility in HTS.
• To demonstrate the application of the assay using a specific GPCR target.

Methods Used

• High-throughput calcium fluorescence assay in 384-well plates.
• Use of CHO-K1 cells expressing the kinin receptor.
• Dual-addition method for simultaneous detection of ligands.
• Optimization of cell density, injection speed, and agonist concentration.

Main Results

• Successful identification of novel small molecule ligands.
• Demonstrated assay reproducibility under optimized conditions.
• Fluorescence signal duration supports the dual addition approach.
• Applicable to various GPCRs that signal through the calcium cascade.

Conclusions

• The dual addition assay is a valuable tool for GPCR ligand screening.
• Optimized conditions enhance assay reliability and efficiency.
• This method can facilitate drug discovery in neuroscience and pharmacology.

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