Cell population dose and dynamic response of ppERK to EGF. A-D. Each panel corresponds to a fixed time after EGF stimulations: 2 min (A), 5 min (B), 10 min (C) and 30 min (D). In each panel, the different colors correspond to different EGF doses as indicated by the visual legend. Each distribution is compiled from 10,000 individual HEK293 cell responses as measured by flow cytometry, and is representative of between three and six independent experiments. Events were gated based on forward and side scatter to exclude debris, dead cells, and cell clusters. The x-axis is the magnitude of activated ERK (ppERK) in arbitrary fluorescence units, and the y-axis is the frequency of observing a particular level of fluorescence in a cell. E. Total ERK abundance data. The best-fit gamma distribution curve is depicted by the green line, while the black line shows experimental data. Data are representative of five independent experiments, and were fit to a gamma distribution. Mean and standard deviation of the fit parameters are k = 5.4; θ = 2.7 × 105 [AU]. F-I. Cells were stimulated with either 0.1 nM (F,H) or 1 nM (G,I) EGF for five minutes, and then analyzed by flow cytometry to measure ppERK and total ERK levels simultaneously. In F-G, black curves correspond to ppERK distributions, and green curves correspond to normalized distributions where ppERK levels in each cell were divided by the total ERK signal intensity in the same cell. To compare the green and black curves on the same axis, intensities for each distribution are divided by their respective mean. In the H-I dot plots, ppERK levels are on the x-axis, whereas total ERK levels are on the y-axis.