| Assay Method Information | |
| | TLR9 Assay |
| Description: | On day 1, 50 μL of each test compound dilution in duplicates or a vehicle control was added to each well of a 96-well plate followed by addition of 150 μL of HEK-Blue hTLR9 cell suspension (1 105 cells/well) and incubated at 37 C./5% CO2 for 2 h. Next, 50 μL of an approximate 3 EC50 concentration of TLR9 agonist, ODN 2006, was added to the wells containing test compounds or the vehicle control. The plates were then incubated at 37 C./5% CO2 for 18 h. For each assay run, vehicle-treated HEK-Blue hTLR9 cells were treated with serial dilutions of agonist to determine EC50 values for the respective run. On day 2, secreted alkaline phosphatase (SEAP) activity was detected in cell culture supernatants. In brief, 30 μL was collected from each well and transferred to a 96-well plate. Next, 200 μL of Quanti-Blue detection reagent was added to each well. Plates were incubated at 37 C. for 60 min. and SEAP activity was assessed by spectrophotometer OD reading at 655 nm. Table A shows the activities of the compounds tested in HEK-Blue hTLR9 cells against ODN 2006. The activities of the compounds against ODN 2006 are presented as IC50 values which were defined as concentrations of the compounds where percent inhibition of the signal induced by agonist is equal to 50%. Exact IC50 values were calculated based on 8-point dilutions for each compound. Approximate IC50 values ( or <) were calculated based on 4-point dilutions for each compound. |
| Affinity data for this assay | |
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