| Assay Method Information | |
| | Determination of agonistic activity of compounds on HEK-Blue hTLR7 cells |
| Description: | 1. HEK-Blue hTLR7 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 into the well plate, so that the final concentrations were 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 h.3. After the incubation, observation under microscope was performed to determine the maximum signal well, the states 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 supermatant was taken and transferred to a new 96-well transparent plate, 90 μL of QUANTI-Blue (Invivogen) detection reagent was added to each well, and incubated at 37° C. for 3 h, 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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