| Assay Method Information | |
| | Determination of Agonistic Activity of Compounds on HEK-Blue hTLR8 Cells |
| Description: | 1. HEK-Blue hTLR8 cells (Invivogen) were cultured in DMEM medium (Hyclone) containing 10% FBS heat-inactivated fetal bovine serum (Corning). On the day of detection, the cell state was observed under a microscope, the cells were collected and resuspended, and the cell concentration was adjusted after counting, 50 μL per well, the total amount of cells was 2×104, and the cells were inoculated in a 96-well plate.2. After the cells adhered to the wall overnight, 50 μL of the test compound at different concentrations was added to the well plate, so that the final concentrations were 100 μM, 25 μM, 6.25 μM, 1.56 μM, 0.39 μM, 0.10 μM, 0.02 μM, 0.006 μM, 0 μM, or 100 μM, 33.3 μM, 11.11 μM, 3.70 μM, 1.23 μM, 0.41 μM, 0.14 μM, 0.05 μM, 0 μM, and the final concentration of DMSO was 1%. The compound was incubated with the cells in a 37° C., 5% CO2 incubator for 20 hours.3. After the incubation, observation was performed under microscope to determine the maximum signal well, the state of the cells with the highest and lowest concentrations of the compound, and whether the compound will crystalize at high concentration. 10 μL of cell culture supernatant was transferred to a new 96-well transparent plate, added with 90 μL of QUANTI-Blue (Invivogen) detection reagent to each well, incubated at 37° C. for 3 hours, and OD620 was read with a multifunctional microplate reader (BMG LABTECH).4. EC50 was calculated by fitting with Graphpad Prism software log(agonist) vs. Response—Variable slope. |
| Affinity data for this assay | |
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