Mitras lb940 plate reader

To quantify Wnt signalling activity in RKO and RKO-APC cells, dual expression of reporter plasmids was used. For this approach, cells were seeded in a 384 well white, flat-bottom polystyrene plate (Greiner) at a density of 7500 cells per well. After 24 h, cells were transfected with 20 ng of actin-renilla and 40 ng of TCF4/Wnt firefly luciferase reporter constructs. Twenty-four hours after reporter plasmids transfection, cells were treated with CHIR99021 (Merck Millipore), XAV939 (Sigma Aldrich) or trametinib (Selleckchem). Reporter levels were measured 48 h after transfection using a Mitras LB940 plate reader (Berthold Technologies) and the TCF4/Wnt-luciferase signal was normalised to actin-Renilla luciferase reporter signal.

The luciferase‐based dual Wnt reporter assay was performed as described previously (Demir et al, 2013 (link)). In brief, cells were seeded in a white, flat‐bottom 384‐ or 96‐well plates. Twenty‐four hours later, cells were transfected with a plasmid encoding tha firefly luciferase under control of a promoter composed by repeats of the TCF4‐binding sites and with a control plasmid encoding renilla luciferase under control of a CMV promoter. Dual‐luciferase readout was performed 48 h after transfection using Mitras LB940 plate reader (Berthold Technologies). The firefly luciferase signal was normalized to the renilla luciferase signal. For RKO three‐independent experiments were performed in total. For HCT116 four‐independent experiments confirmed these results.