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

Cytometry. 2002 Jun 1;48(2):71-9. doi: 10.1002/cyto.10115.


Background: Nuclear factor kappa B (NF-kappaB) is a ubiquitously expressed transcription factor that regulates cytokine and immunoglobulin (Ig) gene expression. In most cell types, the inactive p50/p65 NF-kappaB heterodimer is located in the cytoplasm, complexed to its IkappaB inhibitory unit. Stimulation of cells by various reagents such as bacterial endotoxin or cytokines leads to a dissociation of NF-kappaB from IkappaB and a rapid translocation of free NF-kappaB to the nucleus. The aim of this article is to define optimal conditions for the measurement of NF-kappaB translocation by both confocal microscopy and flow cytometry.

Methods: Four commercial anti-NF-kappaB 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-kappaB 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-kappaB 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-kappaB translocation and can be adapted to identify new molecules that activate dendritic cells.

Publication types

  • Evaluation Study

MeSH terms

  • Animals
  • Cell Nucleus / drug effects
  • Cell Nucleus / immunology
  • Cell Nucleus / metabolism
  • Cell Separation
  • Cells, Cultured
  • Dendritic Cells / drug effects
  • Dendritic Cells / immunology
  • Dendritic Cells / metabolism*
  • Dose-Response Relationship, Immunologic
  • Escherichia coli / immunology
  • Flow Cytometry / methods*
  • Humans
  • Image Cytometry / methods*
  • Lipopolysaccharides / pharmacology
  • Lymphocyte Activation
  • Mice
  • Microscopy, Confocal*
  • NF-kappa B / metabolism*
  • Reproducibility of Results


  • Lipopolysaccharides
  • NF-kappa B