NF-κB is involved in various transcriptional programs induced by variety of cellular processes, such as cell proliferation, survival, differentiation, stresses, and in particular immune and inflammatory responses. And its aberrant regulation is implicated in diverse diseases including cancer, metabolic disorders, autoimmune or inflammatory diseases. Therefore, the signaling pathway leading to the activation of NF-κB is controlled at multiple levels through elaborate positive and negative regulatory components. Further, optimal NF-κB activation is also modulated by post-translational modifications of NF-κB components, including the p65, via the phosphorylation, acetylation, ubiquitination, and so on. Crosstalk with other transcription factors, such as IRF3, STAT3, c-Jun, and c-Fos, can also influence the activity of NF-κB. As a result, direct analysis of the transcriptional activity of NF-κB is indispensable for the study of NF-κB activation pathway.
To this end, we generated stable NKL reporter cell lines that express GFP under the control of NF-κB transcription response element (TRE), instead of transient transfection of reporter vector. We have selected transduced cells with selective antibiotics and further isolated the responding cells by FACS sorting. Measurement of GFP expression following NF-κB-activating stimuli enables direct single cell-based FACS analysis of NF-κB activation, instead of commonly used indirect assays (e.g. phosphorylation of NF-κB components, degradation of IκBα, nuclear translocation and DNA binding activity of p65). For the stimulation of NKL reporter cells, we immobilized NK cell receptor-specific antibodies onto high-binding flat-bottom 96well plate. This protocol describes detailed steps of constructing reporter cells and the analysis of NF-κB transcriptional activity of NK cells following receptor stimulation.