Il 17c hek blue reporter cells

IL-17C HEK-Blue reporter cells (Invivogen) were transfected in 12-well plates with single guide RNA targeting either IL17RA or IL17RB and Cas9 nuclease (Synthego) using Lipofectamine CRISPRMAX Cas9 Transfection Reagent (Thermo Fisher). Five days post-transfection, cells were seeded into 10-cm dishes under limiting dilution. After 2–3 weeks, well-separated single colonies were transferred (via cloning discs) into 24-well plates, then expanded and screened for loss of receptor expression and responsiveness to IL-25.

IL-17C HEK-Blue reporter cells (Invivogen) stably expressing human IL-17RA, ACT1, an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene and endogenous IL-17RB were used to determine NF-κB activity stimulated by IL-25 or IL-17RB mutants. Surface expression of exogenous IL-17RB variants was quantified via epitope staining of the extracellular HA tag and showed overlapping but small differences in expression. The double mutants display greatly reduced signalling efficacy, which was not explained by differences in surface expression. For IL-25 assays, 25,000 cells were seeded overnight into 96-well flat-bottom microplates in the presence of varying doses of IL-25 (WT or mutant). For IL-17RB assays, cells were transfected with plasmid encoding WT or mutant IL-17RB, then seeded into 96-well plates in the presence of WT IL-25 24 h post-transfection. After overnight stimulation, SEAP activity was determined using Quanti-BLUE reagent according to the manufacturer’s directions (Invivogen) and read at an absorbance of 640 nm on a SpectraMax Paradigm plate reader using SoftMax Pro v7.1 (Molecular Devices). Cells were maintained according to manufacturer’s directions.