| Assay Method Information | |
| | In Vitro Receptor Binding Activity of Human Toll-Like Receptor 7 (TLR7) and Human Toll-Like Receptor 8 (TLR8) |
| Description: | 1. The compound was added to a cell plate in a 3-fold gradient, with 10 concentrations (5000 nM, 1667 nM, 556 nM, 185 nM, 62 nM, 21 nM, 6.9 nM, 2.3 nM, 0.76 nM, and 0.25 nM) obtained, and two duplicate wells were set for each concentration. 1 L of DMSO was added to each negative control well. 2. The cells cultured in a T150 flask were taken out from a CO2 incubator, and the cell culture supernatant was discarded. The resulting cells were washed once with Dulbecco s phosphate buffered saline (DPBS). The flask was added with about 10 mL of the culture medium, and tapped to detach the cells. The resulting cell mass was gently pipetted evenly. The cells were counted and the cell suspension was adjusted to 500,000 cells/mL with the culture medium. Then 100 L of diluted cells (50,000 cells/well) were added to each well of a 96-well plate containing the compound. 3. The compound and cells were co-incubated in an incubator at 37 C. with 5% CO2 for 24 hours. 4. Activity assay on the compound: 20 L of the induced cell supernatant from each well was added to a cell culture plate containing 180 L of QUANTI-Blue reagent, and after incubation at 37 C. for 1 hour, the optical density absorbance at 650 nm (OD650) was assayed for each well using a multi-functional microplate reader. 5. Activity assay on the cells: luciferase signal (RLU) was detected using a multi-functional microplate reader as per the process described in the instructions of ATPlite 1 Step. 6. Data analysis: compound activity: OD650 values were analyzed using a GraphPad Prism software and the dose-response curves of the compound were fitted to calculate EC50 values (half maximal effect concentration) for the compound. |
| Affinity data for this assay | |
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