(A) Cells process pathogen signal dose and duration to dynamically activate NF-κB, which induces gene expression and coordinates the innate immune response. We test the role of pathogen load by varying LPS concentration from 0.25 ng/ml to 500 ng/ml using microfluidic cell culture. (B) Time series images of NF-κB activation following LPS treatment. Top row: high LPS dose causes nearly 100% of cells to respond synchronously. Bottom row: at low LPS concentration, less than 5% of cells respond and initiate NF-κB activation with variable, delayed timing. The cells respond digitally, with nearly all cytoplasmic NF-κB moving into the nucleus. The response amplitude (indicated by peak intensity of nuclear p65-dsRed fluorescence) depends on the initial NF-κB abundance in the nucleus and exhibits high variability across doses. (C) Trajectories of NF-κB activation (intensity of nuclear p65-dsRed) tracked in single cells over time for LPS doses ranging from 500 to 0.25 ng/ml. As the LPS dose decreases, response timing becomes delayed and variable, and the percent of responding cells in the population drops. (D) Across the LPS doses tested: top panel, dose-response curve of the fraction of active cells (plotted is the mean of two duplicate cell chambers in the chip for each condition), middle panel, the intensity of nuclear NF-κB at the peak of the response, and lower panel, time until the peak of the response. Peak nuclear NF-κB amplitude is highly variable across doses. The dose response shows a sharp drop in fraction of active cells between 1 and 5 ng/ml concentration, indicating that the activation threshold is within this range for most cells. With lower dose, the response time increases in both median duration and variability. In middle and lower panels, data points and error bars represent median and interquartile range, respectively. (E) NF-κB dependent gene expression dynamics under varied LPS concentrations (blue: 500 ng/ml, green: 100 ng/ml, red: 50 ng/ml). With lower LPS concentration, several genes show delayed induction. TNF dose-modulated expression of the same genes can be found in Figure 3 of Tay et al. (2010) and Figure 2A of Pękalski et al. (2013).