Measurement of nuclear factor-kappa B translocation on lipopolysaccharide-activated human dendritic cells by confocal microscopy and flow cytometry.
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.
bioMérieux-Pierre Fabre, Centre d' Immunologie Pierre Fabre, 5 avenue Napoléon III, 74160 St. Julien en Genevois, France.
Aline
A
Blaecke
Yves
Y
Delneste
Nathalie
N
Herbault
Pascale
P
Jeannin
Jean-Yves
JY
Bonnefoy
Alain
A
Beck
Jean-Pierre
JP
Aubry
D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D004306:Dose-Response Relationship, Immunologic; D004926:Escherichia coli / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation; D051379:Mice; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000378:metabolism*; D015203:Reproducibility of Results
D008070:Lipopolysaccharides; D016328:NF-kappa B