| Assay Method Information | |
| | HEK293 luciferase |
| Description: | Purpose of the AssayThe purpose of this assay is to identify small molecule inhibitors of Keap1, preventing Keap1 binding to Nrf2, and thus activating the Nrf2 signalling pathway in HEK-293 ARE firefly luciferase transiently transfected cells. HEK-293 cells express wild-type Keap1 protein and therefore have low constitutive levels of Nrf2 protein and Nrf2-dependent transcription. The cell line was generated in house by transient transfection and possesses 2× tandem repeats of the ARE transcriptional response element upstream of firefly luciferase. When Keap1 is inhibited, Nrf2 signalling is activated, and there is an increase in luciferase expression and light emission.Assay WorkflowCryopreserved HEK ARE luciferase cells were rapidly thawed in a 37° C. water bath and resuspended in assay culture medium (DMEM, 10% FCS, 2 mM L-glutamine). The cell suspension was pelleted by centrifugation (5 mM; 300 g) using a Heraeus benchtop centrifuge. Supernatant was removed and the pellet was gently resuspended in 5 mL culture medium per vial. Cell viability and cell number was measured using an Invitrogen Countess automated cell counter (typical viability 80%), and cells were diluted to a density of 5.0×105 cells/mL.Cells were then plated out into white 384-well plates (Greiner 781080) using a Multidrop Combi, 20 μL/well, to give 10,000 cells/well. As appropriate, test compounds had been added to wells prior to dispensing cells using an Echo 555 acoustic dispenser (Labcyte). Plates were left at room temperature for 10 min to promote even cell settling across the plate before incubation for 18 h at 37° C., 95% humidity and 5% CO2.After 18 h incubation, plates were removed from the incubator and allowed to cool to room temperature for 30 min, before 10 μL per well SteadyGlo reagent detection reagent was added (as per manufacturer's instructions, 10 min before required 10 mL room temperature buffer was added to one vial of lyophilised substrate and inverted several times to fully dissolve). Plates were then incubated in the dark for 30 min before reading on an Envision plate reader.Preparation of Compounds for ScreeningCompounds were acoustically dispensed using a Labcyte Echo, using a 10 pt dose curve with a top concentration of 100 μM. Assay wells were backfilled with DMSO to a total of 60 nL to maintain 0.3% (v/v) DMSO through the assay.Data Analysis Software and Calculation of ResultsThe Stimulator signal was defined using 30 μM tert-butyl hydroquinone (tBHQ) and the Neutral signal by DMSO vehicle control. All calculations were performed using GeneData. |
| Affinity data for this assay | |
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