High-throughput, single-cell NF-κB dynamics

Abstract

Single cells in a population often respond differently to perturbations in the environment. Live-cell microscopy has enabled scientists to observe these differences at the single-cell level. Some advantages of live-cell imaging over population-based methods include better time resolution, higher sensitivity, automation, and richer datasets. One specific area where live-cell microscopy has made a significant impact is the field of NF-κB signaling dynamics, and recent efforts have focused on making live-cell imaging of these dynamics more high-throughput. We highlight the major aspects of increasing throughput and describe a current system that can monitor, image and analyze the NF-κB activation of thousands of single cells in parallel.

Figure 1. The advantages of using automated, live-cell microscopy to study NF-κB dynamics

Some advantages are biological, such as the ability to detect variation in cell behavior, from larger-scale phenotypic heterogeneity to rare events involving as little as a single cell, and deriving biological parameters based on single-cell measurements. Others are technical, including the ability to perform long experiments at fine time resolution, making several measurements simultaneously and having precise, dynamic environmental control.

Figure 2. Population measurements can obscure heterogeneous single-cell behaviors

The NF-κB response of single cells to a high concentration of TNF-α, in terms of single-cell (top) and population (bottom) level behavior, is shown schematically in (A). At lower concentrations, the response is less pronounced, which can be explained by either of two models. (B) The first model is that all of the cells behave roughly identically and with a lower intensity response. (C) The second model is that some of the cells respond to the stimulus with a normal response (blue), while others do not respond at all (green). These two models are indistinguishable at the population level.

Figure 3. Workflow diagram for high-throughput single-cell imaging of NF-κB dynamics

The steps involved in a live-cell microscopy study of NF-κB are outlined and corresponding difficulties and issues are listed below. Each box is described in more detail in the main text.