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Conditions in terms of cell density, culture medium, and incubation have to be respected. It is very important that there is no Cell Growth Medium in the 96-well plate. Presence of Cell Growth Medium can lead to inappropriate conditioning of the cells and could explain lower signals. Cells in Cell Growth Medium have to be washed in PBS and added at the right cell density in Cell Assay Medium to the 96-well plate. The other factor to take into account is the complete reconstitution of Wnt3a in deionized water. Once reconstituted, Wnt3a can be diluted in Assay Diluent. The optimal concentration of Wnt3a has to be determined by the end user with concentrations ranging from 150 to 500 ng/mL. Incubation of cells with Wnt3a can be increased from 12 hours to 20 hours if necessary. We also suggest the use of black 96-well plate with clear bottom to limit well-to-well cross-talk and improve signal-to-noise ratio. Finally, we would suggest reading the plate 10 minutes after the addition of the luciferase substrate. More than 20-25 minutes and the signal will start to decrease.