NIH3T3 Wnt reporter cell line

Source Mouse fibroblast cell line
Synonyms Wnt reporter, TCF reporter, LEF reporter cell line Background The WNT gene family consists of structurally related genes that encode secreted signaling proteins, membrane bound receptors, and signaling transduction proteins. These proteins have been implicated in oncogenesis, adipogenesis, etc. and in several other developmental processes, including regulation of cell fate and patterning during embryogenesis. Activity of the Wnt signaling pathway leads to nuclear translocation of β-catenin and the formation of TCF transcription factor complex. The TCF complex interacts with Wnt gene transcriptional response elements and leads to the expression of Wnt-responsive genes. Product Description This Wnt reporter cell line is designed to monitor the activity of β-catenin-based Wnt signal transduction pathway. This mouse fibroblast cell line hosts CMV promoter, tandem repeats of the TCF transcriptional response element, and luciferase gene. Activity The luciferase activity from the Wnt reporter cell line increases 25 fold after 8 hours treatment with 10 ng/mL of recombinant mouse Wnt3a.

Handling and Storage The cell line may be shipped in dry ice or RT in either 25 cm2 flask or 15 mL tube. If the cell line is shipped in dry ice, after receiving, store the cells at -80C or in liquid nitrogen or culture under standard culture conditions. Luc Assay Using normal tissue culture plate: Seed 0.5 mL of cells into each well of 24 wells plate (tissue culture-treated) at a density of 10 x 104 cells/mL in complete DMEM medium (Corning Catalog: 15-013-CV plus 2 mM L-glutamine, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate), incubate cell at 5% CO2, 37C incubator overnight, replace complete DMEM with 198 µL DMEM without serum, add 2 µL of control buffer or Wnt3a (concentration range: 0.06 to 1 µg/mL), return plate into 5% CO2, 37C incubator and incubate for 6 to 8 hours, suction out medium, transfer 50 µL cell lysate from each well into the wells of a 96 well black plate, read fluorescent first, and then add 50 µL of Luciferase substrate into each well, read Luciferase activity within 30 min. Fluorescent reading can serve as control of cell numbers.