Quantify GPCR ligand binding in live cells using a Non-Radioactive HTS Format
Alternatively, fluorescent GPCR ligands provide a safer method for determining binding affinities. However, quantification of fluorescent ligand binding tends to rely on time-consuming high resolution fluorescence image capture. This approach can be lengthy and tedious, resulting in 96-well plate read times of 30 to 90 minutes per plate. Thus these High Content Analysis (HCA) assays are too slow to be useful for High Throughput Screening (HTS).
In a new application note, we show how the adjustable focal height feature and the advanced Direct Optic Bottom Reading of the PHERAstar FS plate reader from BMG LABTECH allows for the quantification of fluorescent ligand binding to live, adherent cells in an HTS format. Using CellAurora’s GPCR fluorescent ligands for different GPCRs, IC50 pKi plots for a 96-well microplate were obtained in read times as fast as 2.5 minutes, producing a Z’ factor greater than 0.60. This is in stark contrast to HCA assays, which can take around 60 minutes to achieve the same endpoint.