Assay Method Information

Assay Name:  In Vitro Binding Assay
Description:  I. Master Stock SolutionUnless specified otherwise, the sample compounds were diluted in 100% anhydrous DMSO including all dilutions. The compounds were tested as the citrate salt (1 equivalent per molecule). If the sample compound is provided in a different solvent all master stock dilutions are performed in the specified solvent. All control wells contained identical solvent final concentrations as the sample compound wells.II. Compound Plate AssayThe sample compounds were transferred from a master stock solution into a daughter plate that was used in the assay. Each sample compound was diluted into an assay buffer (1×HBSS with 20 mM HEPES, 2.5 mM Probenecid, and 0.4% Free Fatty Acid BSA) at an appropriate concentration to obtain final specified concentrations.III. Antagonist Assay FormatUsing the EC80 values that were determined real-time, stimulated all pre-incubated sample compounds and reference antagonists (if applicable) were compared with the EC80 values of reference agonist. These were read for 180 seconds using the FLIPRTETRA (This assay added reference agonist to respective wells-then fluorescences measurements were collected to calculate IC50 values).
Affinity data for this assay
 

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