Measurement of nuclear factor-kappa B translocation on lipopolysaccharide-activated human dendritic cells by confocal microscopy and flow cytometry

Abstract

Background: Nuclear factor kappa B (NF-κB) is a ubiquitously expressed transcription factor that regulates cytokine and immunoglobulin (Ig) gene expression. In most cell types, the inactive p50/p65 NF-κB heterodimer is located in the cytoplasm, complexed to its IκB inhibitory unit. Stimulation of cells by various reagents such as bacterial endotoxin or cytokines leads to a dissociation of NF-κB from IκB and a rapid translocation of free NF-κB to the nucleus. The aim of this article is to define optimal conditions for the measurement of NF-κB translocation by both confocal microscopy and flow cytometry. Methods: Four commercial anti-NF-κB antibodies were evaluated by confocal microscopy, after using two methods of fixation and permeabilization of the cells. These antibodies were examined further by flow cytometry on purified nuclei. Results: Paraformaldehyde-methanol treatment of dendritic cells is a good combination to visualize NF-κB translocation by confocal microscopy. Three of the four antibodies tested gave good results on nonactivated and on lipopolysaccharide (LPS)-activated dendritic cells. The measurement of NF-κB translocation by flow cytometry on purified nuclei is a quick and sensitive method. Only one of the four evaluated antibodies showed a significant difference between nonactivated and activated cells. Conclusions: Microscopy and flow cytometry are quick and reproducible methods to measure NF-κB translocation and can be adapted to identify new molecules that activate dendritic cells. © 2002 Wiley-Liss, Inc.