| Assay Method Information | |
| | Evaluation of NLRP3 Inflammasome Inhibitory Activity Assay |
| Description: | The NLRP3 inflammasome inhibitory activity of test compounds were evaluated CM the basis of the inhibitory activity of the IL-1β, production in THP1-Null cells (Product Number: thp-null, InvivoGen). Cells were maintained for culture in RPMI-1640 media containing 10% (v/v) fetal bovine serum, 25 mmol/1. RUES, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/ml normocin, and 200 μg/mL hygromycin B (set at 37° C., 5% CO2/95% air). Cells were suspended with media for assay containing 0.5 μmol/L PMA (RPMI-1640 media containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin), and the suspended cells were seeded on Corning (registered trademark) 384-well Flat. Clear Bottom Black Polystyrene TC-treated Microplates (25,000 cells/25 μL/well)(followed by incubation (set at 37° C., 5% CO2/95% air) overnight supernatant of the culture was removed, and thereto was added media for assay (25 UL/well) containing 1 μg/mL Lipopolysaccharides (Product Number: L2654, Sigma-Aldrich (registered trademark)). Then, the culture was further incubated for 3 hours (set at 37° C., 5% CO2/91% air). The supernatant of the culture was removed. Then, a vehicle solution prepared from Opti-MEM (trademark) medium (Product Number: 31985-070, Invitrogen) was added to blank-setting wells and control-setting wells (20 μL/well), followed by incubation for 15 minutes (set at 37° C., 5% CO2/91% air). A solution containing a test compound (20 μL/well) was added to test compound-setting wells. Further, Opti-MEM (trademark) medium containing Nigericin (Product Number: N7143, Sigma-Aldrich (registered trademark)) was added to the control-setting wells and test compound-setting wells (5 μL/well), followed by incubation for 1.5 hours (set at 37° C., 5% CO2/95% air). The final concentration of Nigericin was adjusted to be 7.5 μmol/L, 5 μL/well of Opti-MEM (trademark) medium was added to the blank setting wells. The supernatant of the culture was cryonically stored (set at −20° C.) until measurement of IL-1β. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |