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Experimental Work Flow


The study of NF-κB activation and localization dynamics is becoming an argument of increasing interest in system biology. Live single-cell imaging is one of the main tools used to study these dynamics.

Traditional biochemical assays like electrophoretic mobility shift assay are able to measure only nuclear NF-κB on a population-level, while live cell imaging permits to analyze the overall level of NF-κB (both active-state in the nucleus and inactive-state in the cytoplasm) and to find differences in a population with the resolution of a single cell. .

The visualization of NF-κB activation is mainly conducted with a fluorescent tag on p65/RELA NF-κB subunit obtained with either transfection or viral transduction. Fluorescence microscope with integrated incubation system for maintaining cell viability are used to image unstimulated or stimulated cells in time-lapse experiments. The information on p65 intensity is obtained with image segmentation. A prior cellular segmentation to track the cells nuclei is done to distinguish and quantify p65 relative fluorescent intensity in the nucleus and in the cytoplasm of the cells. For this reason, nuclei needs to be marked in a different fluorescent channel, using nuclear dyes like Hoechst stain or fluorescent histones. The quantification of p65 intensity has been described as ratio of nuclear to cytoplasmic intensity, nuclear to total intensity , or an average value.

Current Work
A pioneering study on NF-κB dynamics was conducted with live cell imaging on SK-N-SH and HeLa cells stable transfected with a plasmid vector expressing for p65 fused with DsRed protein, suggesting an oscillatory dynamics of NF-κB from cytoplasm to the nucleus and viceversa, due to feedback loop regulation related to the NF-κB inhibitor IκBα.

The behavior of these cells treated with different short pulses of inflammatory stimulation of TNF-α gave raise to different patterns of NF-κB dependent gene expression, supporting the idea that oscillatory dynamics has a role in genetic transcription.

Plasmid vector for GFP-tagged p65 have also been used to study NF-κB dynamics in HeLa cells. Combining live cell imaging and target gene transcription in the same cells, it was shown that nuclear NF-κB levels are very heterogeneous from cell to cell and TNF-induced transcription correlates best with fold change in nuclear NF-κB than its absolute nuclear abundance.

In addition to TNF-α concentration also duration of the stimulation plays a fundamental role and can shape cell fate decisions. .

Single-cell level analysis of NF-κB activation in presence of lipopolysaccharide also showed heterogeneous response compared to previous population-level studies.

To avoid interference between endogenous and exogenous p65 subunit, a EGFP/RelA knock-in mouse has been generated, to strictly rely on endogenous level of the protein. This animal model has been successfully applied in recent studies.

Finally, to make live cell imaging more high-throughput, microlfuidic platform for cell culture stimulation enriched the study of NF-κB in living cells by imposing time-varying stimuli similar to the ones seeing by cells in their microenvironment. With the combination of both live single-cell imaging and microfluidics, it was found that in mouse fibroblasts NF-κB dynamics are synchronous with oscillating TNF external stimulation and becoming entrained. On the contrary, similar cells showed damped oscillation where the oscillatory peaks tend to decrease.