Cayman’s reverse transfection reporter assays have overcome many of the disadvantages of other transfection approaches. In this method, a proprietary transfection complex containing DNA and an optimized mixture of lipids and proteins has been evenly immobilized on the culture surface of multi-well plates. Adherent cells, supplied by the user, are applied directly to the plate and allowed to grow in the coated wells. Using this method, the uptake of the DNA complex by the cell is increased dramatically compared to solution-phase transfection, enhancing both the transfection efficiency and co-transfection efficiency for multiple plasmids. Cayman’s Bile Acid Nuclear Receptor FXR (NR1H4) Reporter Assay Kit consists of a 96-well plate coated with a transfection complex containing DNA constructs for expressing FXR and a BSEP promoter-regulated secreted alkaline phosphatase (SEAP) reporter (FXR Reverse Transfection Strip Plate). Cells grown on the transfection complex will express FXRa2 inside the cells within 24 hours from an engineered plasmid construct. Binding of agonists to FXR triggers its transcription factor activity. Activation of the BSEP promoter on the reporter construct by FXR results in the expression of SEAP, which is secreted into the cell culture medium. Aliquots of media are collected 6-24 hours after stimulation, and SEAP activity is measured following addition of a luminescence-based alkaline phosphatase substrate (SEAP Substrate (Luminescence)). The kit is easy to use and can be readily applied to high-throughput screening for therapeutic compounds regulating the activation of FXR. A selective synthetic agonist, XL335, is included in the kit for use as a positive control.{37177} The kit provides sufficient reagent to measure SEAP activity at three time points using the three included assay plates.
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