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| | GPR142 Agonist Effect as Measured by IP-1 Assay |
| Description: | HEK293 cells expressing human GPR142 are maintained in DMEM supplemented with 10% FBS and 800 μg/ml G418 (Geneticin) at 37° C. and 5% CO2. The cells are plated in 384 well plates at 5000 cells per well and allowed 18 hours for attachment. After addition of compounds at varying concentrations ranging from 30 μM to 1 nM, cells are incubated for 1 hr. IP-1 measurements are performed using an IP-One HTRF assay kit (Cisbio) according to manufacturer's protocol using assay buffer containing 1×HBSS (+Ca, +Mg), 0.1% BSA, 50 mM LiCl and 20 mM HEPES, pH 7.2. The reaction is stopped by addition of IP1-d2 (IP-1 coupled to an organic HTRF acceptor) followed by cryptate solution (http://www.htrf.com/usa/htrf-chemistry). The plates are incubated at 25° C. for 1 hr. Fluorescence is read in an Envision instrument at 665 nm and 620 nm wavelength. The ratio of 665 nm/620 nm is calculated and converted to IP-1 levels using an IP-1 standard curve. The data is fit to a 4 parameter-fit logistics to determine EC50 values. |
| Affinity data for this assay | |
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