Measurement of NF-κB Activation in TLR-Activated Macrophages.

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.