Blaecke Aline, Delneste Yves, Herbault Nathalie, Jeannin Pascale, Bonnefoy Jean-Yves, Beck Alain, Aubry Jean-Pierre
bioMérieux-Pierre Fabre, Centre d' Immunologie Pierre Fabre, 5 avenue Napoléon III, 74160 St. Julien en Genevois, France.
Cytometry. 2002 Jun 1;48(2):71-9. doi: 10.1002/cyto.10115.
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.
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.
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.
核因子κB（NF-κB）是一种广泛表达的转录因子，可调节细胞因子和免疫球蛋白（Ig）基因的表达。在大多数细胞类型中，无活性的p50/p65 NF-κB异二聚体位于细胞质中，与它的IκB抑制单位结合。用各种试剂如细菌内毒素或细胞因子刺激细胞会导致NF-κB与IκB解离，游离的NF-κB迅速转位至细胞核。本文的目的是确定通过共聚焦显微镜和流式细胞术测量NF-κB转位的最佳条件。
在使用两种细胞固定和通透方法后，通过共聚焦显微镜评估四种商用抗NF-κB抗体。在纯化的细胞核上通过流式细胞术进一步检测这些抗体。
用多聚甲醛-甲醇处理树突状细胞是通过共聚焦显微镜观察NF-κB转位的良好组合。所测试的四种抗体中的三种在未活化和脂多糖（LPS）活化的树突状细胞上均产生了良好的结果。通过流式细胞术在纯化的细胞核上测量NF-κB转位是一种快速且灵敏的方法。所评估的四种抗体中只有一种在未活化和活化细胞之间显示出显著差异。结论：显微镜和流式细胞术是测量NF-κB转位的快速且可重复的方法，可用于鉴定激活树突状细胞的新分子。
