(A) Time-course of the 561/640 ratio measured in serum-starved EE7 cells grown in 96-well plates. The cells were labeled with MG-Bis-SA and incubated with 1, 4 or 20 ng/ml EGF at 37°C. The 561/640 ratio was measured as described in ‘Material and methods’. (B) To test the robustness of the assay at physiologically relevant EGF concentrations, we calculated the Z’-factor from the assay run with half a plate run as control and the other half with the addition of 1, 2 or 4 ng/ml EGF. The Z’-factor was calculated to be 0.76–0.88, indicating the high robustness of the assay. (C) EE7 cells grown in 96-well plates were serum-starved, incubated with vehicle (DMSO) or PD158780 (50 nM) for 30 min, labeled with MG-Bis-SA and further incubated with the range of EGF concentrations for 15 min at 37°C in the same media. The 561/640 ratio was measured as described in ‘Material and methods’.