Measurement of NF-κB Activation in TLR-Activated Macrophages

  • Orna Ernst
  • Sharat J. Vayttaden
  • Iain D. C. FraserEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1714)


Nuclear factor kappa-B (NF-κB) is a key transcription factor in the regulation of the innate immune inflammatory response in activated macrophages. NF-κB functions as a homo- or hetero-dimer derived from one or more of the five members of the NF-κB family, and is activated through a well-studied process of stimulus-dependent inhibitor degradation, post-translational modification, nuclear translocation, and chromatin binding. Its activity is subject to multiple levels of feedback control through both inhibitor protein activity and direct regulation of NF-κB components. Many methods have been developed to measure and quantify NF-κB activation. In this chapter, we summarize available methods and present a protocol for image-based measurement of NF-κB activation in macrophages activated with microbial stimuli. Using either a stably expressed GFP-tagged fusion of the RelA NF-κB protein, or direct detection of endogenous RelA by immunocytochemistry, we describe data collection and analysis to quantify NF-κB cytosol to nuclear translocation in single cells using fluorescence microscopy.


NF-κB RelA Nuclear translocation Macrophage Transcription factor GFP High-content imaging 



We thank Dr. Jing Sun for helpful discussions and comments on the nuclear translocation protocols. This work was generously supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases.


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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Orna Ernst
    • 1
  • Sharat J. Vayttaden
    • 1
  • Iain D. C. Fraser
    • 1
    Email author
  1. 1.Signaling Systems Unit, Laboratory of Immune System BiologyNational Institute of Allergy and Infectious Diseases, National Institutes of HealthBethesdaUSA

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