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Detecting Signaling in Single Cells

Combining high-throughput immunofluorescence with microfluidics allows signaling dynamics to be quantified in single cells or across populations. Signaling events are dynamic, and cells may exhibit individual variation that is masked by analysis of populations of cells. These properties make it difficult to fully evaluate cell signaling by conventional biochemical techniques, and thus there is a need for techniques and systems that enable researchers to probe single cell responses. Cheong et al. report the development of a microfluidics device called Imstain, which can be combined with immunocytochemistry to allow the rapid, precise delivery of reagents, as well as the power of immunofluorescence to track changes in subcellular localization. With this device, they demonstrate that the microfluidic experiments had much lower variability in cell density and in responses in replicate samples than did experiments performed in multiwell plates. They explore the cellular response to tumor necrosis factor–α (TNF-α) and show that a putatively selective inhibitor of nuclear factor κB (NF-κB) that targets the inhibitor of κB kinase (IKK) also decreases the activity of the transcription factor AP-1 that is activated by c-Jun N-terminal kinase (JNK). Several explanations are possible for this result: (i) the inhibitor also inhibits enzymes that participate in the JNK arm of the response; (ii) NF-κB signaling feeds back on TNF-α, and in its absence there is less total stimulation, thus less AP-1 activity; or (iii) IKK may participate in AP-1 activation through an as-yet-uncharacterized mechanism. Although Cheong et al. do not further explore this question, it proves that their device can lead to hypothesis generation. They also explored the kinetics of NF-κB activation and found that repeated stimulation with TNF-α resulted in a lower-amplitude response to each subsequent exposure, suggesting an adaptation or down-regulation of the response. Single-cell analysis of NF-κB activity revealed that, unlike cells overexpressing a green fluorescent protein–tagged form of the p65 subunit of NF-κB, nuclear translocation of p65 in single cells reflected the behavior of the population and individual cells did not exhibit asynchronous spiking. Thus, application of microfluidics technology has the potential to reveal new biology, improve reproducibility and experimental accuracy while saving time and cost, and drive the generation of testable hypotheses. R. Cheong, C. J. Wang, A. Levchenko, High-content cell screening in a microfluidic device. Mol. Cell. Proteom. Papers in Press, published 24 October 2008 as 10.1074/mcp.M800291-MCP200. [PubMed]