Activation of nuclear factor kappa B (NF-?B) assayed by laser scanning cytometry (LSC)

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Abstract
Nuclear factor kappa B (NF‐κB)/rel is the family of ubiquitous transcriptional activators involved in regulation of diverse immune and inflammatory responses. It also plays a role in control of cell growth and apoptosis. In its inactive form NF‐κB remains in the cytoplasm sequestered through interaction with IκB protein. Rapid translocation of NF‐κB from cytoplasm to nucleus that occurs in response to extracellular signals is considered to be a hallmark feature of its activation. The translocation of NF‐κB in HL‐60, U‐937 and Jurkat leukemic cells as well as in human fibroblasts induced by tumor necrosis factor α (TNF‐α) or phorbol myristate acetate (PMA) was presently measured by laser scanning cytometry (LSC). NF‐κB was detected immunocytochemically with FITC‐tagged antibody and its presence in the nucleus vis‐a‐vis cytoplasm was monitored by measuring the green fluorescence integrated over the nucleus, which was counterstained with propidium iodide (PI), and over the cytoplasm, respectively. Activation of NF‐κB led to a rapid increase in NF‐κB‐associated fluorescence measured over the nucleus (FN) concomitant with a decrease in fluorescence over the cytoplasm (FC), which was reflected by an increase in FN/FC ratio. This rapid assay of NF‐κB activation can be combined with morphological identification of the activated cells or with their immunophenotype. Bivariate analysis of NF‐κB expression versus cellular DNA content makes it possible to correlate its activation with the cell cycle position. The described method has a potential to be used as a functional assay to monitor intracellular translocation of other transcriptional activators such as p53 tumor suppressor protein or signal transduction molecules. Cytometry 33:376–382, 1998.