Tuesday, September 8, 2009

A Cost-Effective Automated Workflow for High-Throughput Screening of G-Protein-Coupled Receptors (GPCRs) Using Fluorescence

Drugs targeting members of the GPCR superfamily represent the core of modern medicine. They account for the majority of the best-selling drugs and roughly 40% of all prescription pharmaceuticals on the market today. The new fluorescent probe for drug discovery FluoForte™, which is provided to cells as a cell membrane-permeable acetoxymethyl (AM) ester, is used to track calcium flux as part of the signal transduction process initiated by GPCR binding. Media is removed and the working dye solution is added using an EL406 Combination Washer Dispenser. Once within the cells, the probe is hydrolyzed by intracellular esterases, leading to the generation of a cell membrane impermeable form, in an analogous manner as commonly used Fluo-3 or Fluo-4 dyes. Intracellular calcium binding to the fluorescent probe increases its fluorescence, which is readily measured using a Synergy Mx reader equipped with a dual-reagent dispenser. This homogeneous fluorescence-based assay workflow is suitable for monitoring calcium mobilization across a broad spectrum of biological targets. The easy-to-use automation protocol does not require the exogenous addition of a quencher dye, which could adversely affect receptor-ligand interaction kinetics. Dye loading in live cells can be performed at 37°C or room temperature, for easier automation. The assay can be performed in a convenient 96-well or 384-well microplate format. The ease-of use of the kit, affordability and high performance of the instruments and minimal installation requirements make the workflow readily accessible, even to academic research groups with modest consumable and instrumentation budgets.

BioTek offers several different instruments that can be used for low cost GPCR screening. The Precision Microplate Sample Processor can be used for drug serial dilution, plate to plate transfer and cell seeding. As mentioned previously, the EL406 can be used to both aspirate media and dispense loading dye. Alternatively, these tasks can be performed using separate instrumentation such as the ELx405 Washer and the MicroFlo Select dispenser. These modular instruments can automate many of the tasks necessary to perform low to medium throughput GPCR screening assays at a reasonable price.

ATP Dose Response in CHO-M1 Cells

Figure 1. ATP Dose Response in CHO-M1 Cells

By: BioTek Instruments

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