| Assay Method Information | |
| | In Vitro Assay for Determining IC50 |
| Description: | Compounds were prepared in DMSO (stock concentration: 1 mM) and 3×serially diluted (10 concentrations) in 384-LDV plate (Labcyte). Ninety nL of serially diluted compounds were transferred from 384-LDV plate into compound plate (PerkinElmer) by Echo 550 (Labcyte), and 30 uL assay buffer (1×HBSS with 20 mM HEPES, pH 7.4, Sigma) were added to each well. For FLIPR assay, culture media was removed and 20 uL of 1× loading dye (Assay buffer with 2 μM Fluo-8 AM, AAT Bioquest; 1 mM Probenecid and 0.0025% pluronic F-127, Sigma) was added to each well. The plate was incubated at 37° C. for 1 hr (avoid light exposure). For FLIPR assay, Excitation wavelength was set at 470/495 nm and Emission wavelength was set at 515/575 nm (Molecular Devices). Assay was performed in both agonist and antagonist modes. For agonist mode, 10 μL of diluted compounds were transferred to cell culture well and incubated at room temperature for 10 min. |
| Affinity data for this assay | |
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