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The Cignal Reporter Assays (GFP) 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. GFP expression is quantitated using a flow cytometer, fluorescent microscope, or fluorometer. The change in the activity of each signaling pathway is determined by comparing the GFP activities 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 from the constitutively expressing CMV-GFP reporter.
Excellent signal to noise ratio
Fluorescence Microscopy: Cignal NFκB
GFP reporter showed that Human Tumor Necrosis Factor Alpha (hTNFα)
activated the NFκB
signaling pathway
293-H cells were transfected with the Cignal NFκB-GFP
reporter or negative 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 10 ng/ml hTNFα. After 18 hours of treatment,
bright field and fluorescent images were taken of the cultures transfected
with the negative control reporter (A and B, respectively) and the Cignal NFκB-GFP reporter (C and D, respectively).
Quantitative Fluorometry: Cignal NFκB
GFP reporter showed that Human Tumor Necrosis Factor Alpha (hTNFα)
activated the NFκB
signaling pathway
293-H cells were transfected with the Cignal NFκB-GFP
reporter or negative 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 10 ng/ml TNFα. After 18 hours of treatment, medium
was removed from the wells and fluorescence activity was measured using a
fluorometer. The fluorescent activity present in treated and non-treated
negative control wells was subtracted from the fluorescent activity in
treated and non-treated Cignal NFκB-GFP reporter wells and relative
fluorescence activities are expressed as arbitrary units. Experiments were
done in triplicates, and the standard deviations are indicated.
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