The Cignal Reporter Assays (luc) include pre-formulated, transfection-ready reporter, negative control, and positive control. The transcription factor reporter and negative control are transfected and subjected to experimental treatments, in parallel. Dual-luciferase results are calculated for each transfectant. The change in the activity of each signaling pathway is determined by comparing the normalized luciferase activities of the reporter in treated versus untreated transfectants. The identically treated negative control transfectants serve as a specificity control. The positive control serves as a control for transfection efficiency, by monitoring GFP expression, as well as a positive control for both the firefly and Renilla luciferase assays.
Average maximum response rate = 857
Average Z' factor at maximum response rate = 0.87
Average coefficient of variation (CV%) = 7.6%
Excellent signal to noise ratio
Cignal LEF/TCF reporter assay showed upregulation of Wnt signaling activity: 293-H cells were transfected with LEF/TCF reporter, negative control and positive control (for transfection protocol refer our user manual). After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 24 hours of transfection, cells were treated with either 400 ng/ml of recombinant mouse Wnt3a protein (mWnt3a) for 18 hours, or 14 hours with 10mM LiCl followed by 4 hours with 400 ng/ml of mWnt3a. Dual Luciferase assay was performed 42 hours after transfection, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated. Cignal LEF/TCF reporter assay measured 857 fold increase in β-catenin activity and, in turn, showed upregulation of Wnt signaling activity by 50 mM LiCl.
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