Cells operate in ever changing environments using extraordinary communication capabilities that emerge from the interactions of genetic circuitry. The mammalian immune response is a striking example of the coordination of different cell types. Cell-to-cell communication is primarily mediated by signaling molecules that form spatiotemporal concentration gradients, requiring cells to respond to a wide range of signal intensities. Here we use high-throughput microfluidic cell culture, quantitative gene expression analysis and mathematical modeling to investigate how single mammalian cells respond to different concentrations of the signaling molecule TNF-α, and relay information to the gene expression programs via the transcription factor NF-κB. We measured NF-κB activity in thousands of live cells under TNF-α doses covering four orders of magnitude. We find, in contrast to population studies, that the activation is heterogeneous and is a digital process at the single cell level with fewer cells responding at lower doses. Cells also encode a subtle set of analog parameters to modulate the outcome; these parameters include NF-κB peak intensity, response time and number of oscillations. We developed a mathematical model that reproduces both the digital and analog dynamics as well as the most gene expression profiles at all measured conditions, constituting a broadly applicable model for TNF-α induced NF-κB signaling in various types of cells. These results highlight the value of high-throughput quantitative measurements at the single-cell level in understanding how biological systems operate.
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